brain inflammatory brain changes in lyme borreliosis · 2010-09-14 · brain (1996), 119,2143-2154...

Brain (1996), 119,2143-2154

Inflammatory brain changes in Lyme borreliosisA report on three patients and review of literature

J. Oksi,1-5'7 H. Kalimo,2 R. J. Marttila,3 M. Marjamaki,2 P. Sonninen,4 J. Nikoskelainen1 andM. K. Viljanen6-7

Departments of internal Medicine, 2Pathology, ^Neurology Correspondence to: J. Oksi, Turku University, Departmentand ^Radiology, Turku University Central Hospital, of Medical Microbiology, Kiinamyllynkatu 13, FIN-205205Department of Medical Microbiology, Turku University, Turku, Finland^National Public Health Institute, Department in Turku and1Turku Immunology Centre, Turku, Finland

SummaryDespite a rapid increase in the number of patients withLyme neuroborreliosis (LNB), its neuropathological aspectsare poorly understood. The objective of this studv wasevaluation of neuropathological, microbiological, andmagnetic resonance imaging (MRI) findings in three patientswith the Borrelia burgdorferi infection and neurologicaldisease from whom brain tissue specimens were available.Perivascular or vasculitic lymphocytic inflammation wasdetected in all specimens. Large areas of demyelination inperiventricular white matter were detected histologically andby MRI in one patient. The disease had a fatal outcome inthis patient. Brain MRI suggested malignancies in two

patients before histopathological studies were carried out.One of these two patients was a child with sudden hemiparesis.Another was a 40-year-old man presenting with epilepticseizures and MRI-detected multifocal lesions, whichdisappeared after repeated courses of antibiotics. Weconclude that cerebral lymphocytic vasculitis and multifocalencephalitis may be associated with B. burgdorferi infection.The presence of B. burgdorferi DNA in tissue samplesfrom areas with inflammatory clianges indicates that directinvasion ofB. burgdorferi may be the pathogenetic mechanismfor focal encephalitis in LNB.

Keywords: Borrelia burgdorferi; Lyme disease; neuroborreliosis; neuropathology; vasculitis

Abbreviations: BBB = blood-brain barrier; ESR = erythrocyte sedimentation rate; H and E = haematoxylin and eosin;HSV = herpes simplex virus; LNB = Lyme neuroborreliosis; PCR = polymerase chain reaction

IntroductionThe meninges, spinal nerve roots and cranial nerves are mostcommonly involved in LNB. Chronic CNS involvementof LNB may mimic diseases such as neurosyphilis,meningoencephalitis of viral, fungal or mycobacterial origin,multiple sclerosis, brain tumor, autoimmune disease, strokeor Alzheimer's disease.

Despite a rapid increase in the number of patients withLNB its neuropathological aspects are poorly understood(Garcia-Monco and Benach, 1995). In principle, the brainlesions can be caused either directly by the spirochaete orindirectly via biologically active substances secreted by hostcells on stimulation with the spirochaete. Borrelia burgdorferiis able to adhere to and penetrate through the endotheliumof CNS blood vessels and then adhere to the glial cells(Garcia-Monco et ai, 1989; Comstock and Thomas, 1989;

© Oxford University Press 1996

Szczepanski et al., 1990). This invasion can already occurat early stages of the infection, even without signs ofinflammation in the CSF (Garcia-Monco et al., 1990; Luftet al., 1992). After invasion of the CNS, the spirochaete maycause direct damage to oligodendroglial cells, which maylead to demyelination (Baig et al., 1991). However, there isincreasing evidence that inflammatory changes in CNS bloodvessels, such as diffuse or focal vasculitis or cerebrovascularinjury, may be an important factor in the development of theCNS lesions and dysfunction in LNB (Midgard and Hofstad,1987; Uldry et ai, 1987; Weder et al., 1987; Wokke et al.,1987). It is conceivable that Lyme borreliosis may also causevascular damage in the CNS, because it is known to causevasculitis and perivascular inflammation in several otherorgans outside the CNS (Johnston et ai, 1985; Camponovo

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and Meier, 1986; Duray, 1989a, b; Meurers et al., 1990;Moody et al, 1990a; Olson and Esterly, 1990; Smith et al.,1991; Karma et al, 1995). The factors involved in thedevelopment of the vascular changes in LNB are not known.It is evident that the spirochaete occurs in very low numbersespecially in the CNS, since its presence in brain tissue hasbeen a rare finding (MacDonald and Miranda, 1987; Weberet al., 1988; Pachner et al., 1989; Kuntzer et al., 1991;Millner et al., 1991; Miklossy et al., 1994), suggesting thatCNS lesions are also caused by inflammatory mediators andnot only by the direct action of the spirochaete itself. Onerecent study showed that B. burgdorferi is capable of directactivation of vascular endothelium promoting recruitment ofleucocytes to perivascular tissues (Sellati et al, 1995).

Modern imaging techniques, such as CT and MRI, areused successfully for the detection of vascular lesions in theCNS of LNB patients (Uldry et al, 1987; Kohler et al.,1988; Belman et al, 1992), and together with sensitive geneamplification methods, such as polymerase chain reaction(PCR), they have improved the possibilities of studying therole of B. burgdorferi in the development of vasculitic lesions.

We describe three patients with CNS lesions shown byMRI and neurological symptoms. In two of the patients,B. burgdorferi DNA was detected by PCR in specimensfrom inflammatory CNS lesions; it was cultivated fromthe CSF of one of these two. In the child with hemiparesisand vasculitis in the brain, borrelia PCR yielded positiveresults with CSF.

Patients and methodsPatientsTwo patients living in an area where Lyme borreliosis isendemic were examined at the Turku University CentralHospital. One patient, who had briefly visited an endemicarea, was examined at the Oulu University Central Hospital.

Magnetic resonance imagingAll patients were examined using a high-field magnet (1.5Magnetom, Siemens) with T2 and T, sequences (TR 2500,TE 90 and TR 600, TE 15). Gadolinium enhancement wasalso used. Axial, coronal, and sagittal planes were imaged.

Assessment of borrelia antibodiesThe IgM and IgG antibodies against sonicated B. burgdorferiwere measured by an in-house ELISA using B. burgdorferisensu stricto (B31, ATCC 35210) as the antigen (Viljanenand Punnonen, 1989). All steps of the ELISA were carried outautomatically with an Auto-EIA II instrument (Labsystems,Helsinki, Finland). Serum samples were tested at a dilutionof 1:100. The results were expressed as relative ELISA units.Seropositivity was determined by comparing antibody resultsfrom test serum samples with those of 110 healthy controls.

The cut-off value for weakly positive results was themean + (2XSD) of the controls. The IgM and IgG antibodiesagainst flagellin were measured using a commercial ELISAtest (Lyme Borreliosis ELISA Kit, 2nd generation; DAKOA/S, Glostrup, Denmark). The CSF samples were tested at adilution of 1:10. The CSF IgM and IgG antibodies againstsonicated B. burgdorferi were measured using the in-houseELISA (EIU units > 10 were considered positive) and againstflagellin by a commercial ELISA (DAKO).

Culture of B. burgdorferiThe specimens (biopsy, CSF or blood) were inoculated intotubes containing BSK-II medium and incubated at 30°C. Thetubes were examined macroscopically twice weekly andpassaged once weekly for at least two months. Dark-fieldmicroscopy was carried out if the colour of the culturemedium indicated growth. The final identification of culturedspirochaetes was based on PCR.

Extraction of DNA for the PCRA 1 ml sample (i.e. minced biopsy specimen, plasma, serumor CSF) was centrifuged (Eppendorf Microfuge, 13000 r.p.m.,10 min), 800 u.1 of the supernatant was removed, and theremaining 200 p.1 was mixed with 300 (il of sodium dodecylsulphate (SDS) solution (0.1 M NaOH, 2 M NaCl and 0.5% ).After incubation at 80°C for 15 min, 200 |il of 0.1 M Tris-HC1 (pH 8) was added. After the SDS treatments, DNA wasextracted with phenol-chloroform, precipitated with ethanoland finally dissolved in Tris-EDTA buffer (Hance etal, 1989).

Polymerase chain reactionA 5 u.1 volume of extracted DNA was added into thereaction tube. The specific targets chosen for the PCRs wereDNA fragments from the flagellin gene sequence of B.burgdorferi. The PCR from all specimens obtained fromPatient 1, and from the brain tissue specimens of Patient 2,was run in two steps, first with external primers prB31/41 —4 and prB31/41-5, resulting in a 730-bp PCR product (Wallichet al, 1990) and then with nested primers WK1 and WK2,resulting in a 290-bp fragment (Kriiger and Pulz, 1991).Finally, the PCR from specimens obtained from Patient 3,and the plasma of Patient 2 was run as described earlier withprimers WK1 and FL7, resulting in a 497-bp PCR product(Kriiger and Pulz, 1991; Picken, 1992; He et al, 1994).Each PCR run included a positive control containing DNAextracted from a reference strain (B31) of B. burgdorferisensu stricto (ATCC 35210). Control brain tissue sampleswere included in the PCR runs in a blinded manner and withnegative results. Every sixth tube of each run was used as anegative control subjected to all above sample treatments.The negative controls remained negative in each run. Onehundred blood donors living in the Turku area providedcontrol samples for the PCR assay with primers WK1 and

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Table 1 Laboratory results of borrelia tests, treatments for LNB, and outcome of three patients with Lyme borreliosisassociated with CNS inflammatory changes

Patient number

Serum antibodiesLymphocyte proliferationCSF antibodiesCSF cultureCSFPCRBlood culturePlasma PCRBone marrow PCR

Brain tissue PCR

Number of treatments for LNBInterval between onset of symptoms and first treatment for LNB (months)Persistence of PCR positivity after onset of antibiotic treatment (months)Outcome

+ = positive; — = negative; ND = not done; A = asymptomatic; B = significantly better than before treatment; D = dead.

1

-

—

+

++

2>39

8D

2

+ +

—-

—

21

16A

3

NDNDND

NDNDND

130B

FL7: one of their samples was positive. Similar studies onblood donors was not done for the other above mentionedPCR assays with different primer pairs. The sensitivity ofthe PCR with WK1 and FL7 primers (He et ai, 1994) wasfound to between 10 and 100 B. burgdorferi cells per reaction.The sensitivities of the other PCR modifications were at thesame level as that with WK1 and FL7 primers. All PCRsused were found highly specific for B. burgdorferi s.l. OtherBorrelia species (B. hermsii, B. parkeri and B. turicatae)and treponemes (Treponema denticola, T. pectinovorum,T. socranskii and T. vincentii) gave negative results.

NeuropathologySurgical samples were fixed in 4% phosphate bufferedformaldehyde and routinely processed into paraffin sections.Brains from autopsies were also fixed in the same fixative.During cutting of the brains, samples from areas judged asabnormal on brain imaging and from areas with macroscopicpathological features were collected and processed intoparaffin sections. The specimens were stained usinghaematoxylin and eosin (H and E), and Luxol Fast Blue-Cresyl Violet stains. Inflammatory cells were demonstratedimmunohistochemically using mouse monoclonal antibodyto CD 45 and leucocyte common antigen (Dakopatts A/S,Glostrup, Denmark). Bound primary antibodies were detectedapplying an appropriate Vectastain (Vector Laboratories,Burlingame, Calif., USA) avidin-biotin-peroxidase detectionkit with diaminobenzidine as chromogen.

ResultsIn two cases (Patients 1 and 2), the brain tissue specimenfrom areas with vasculitic lesions contained DNA of B.burgdorferi as evidenced by PCR amplification (Table 1).In one of them, culture as well as PCR of the CSF

showed B. burgdorferi (Patient 1). In addition, the plasmaof both of the patients (Patients 1 and 2) contained DNAof B. burgdorferi. In the remaining one patient, DNA of B.burgdorferi was amplified in the CSF (Patient 3). In allpatients, a positive PCR result was obtained from more thanone specimen taken and analysed at different times (Table1). Two of the patients had antibodies against B. burgdorferiin the serum but none of the patients had them in the CSF.Circulating immune complexes were found in both twopatients studied (Table 1).

In one patient (Patient 1), MRI of the brain showed largeperiventricular lesions in white matter and the disease had afatal outcome. MRI findings in two patients (Patients 2 and3) suggested malignancy before the histopathological studieswere carried out. One of them was a child with suddenhemiparesis. Another was a 40-year-old man who presentedwith epileptic seizures; his MRI showed multifocal lesions,which disappeared after repeated antibiotic therapy.

Perivascular or vasculitic lymphocytic inflammation in theclinical or autopsy brain biopsy specimens was detected in allthree patients. Large areas of demyelination in periventricularwhite matter were detected in one patient (Patient 1). Detailedneuropathological descriptions are included in the followingcase reports.

Patient 1The patient was a 51-year-old woman with a history ofprogressive lymphedema of the left lower limb since 1954.She had suffered from erysipelas in the left lower leg anderythema nodosum in both legs, and had also had recurrentfever episodes several times a year. Lung fibrosis, heartinsufficiency and chest pain atypical of coronary heart diseasedeveloped at the age of 30-35 years. She had received long-term corticosteroids and several courses of antimicrobialdrugs.

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In 1985, the patient had a 3 week period of fever and facialredness suggestive of lupoid erythema. Despite corticosteroidtreatment, a spiking fever persisted. At hospital, no infectionfocus was found. Antimalarial drugs combined withmethylprednisolone were given for two years, but episodes ofmild fever reappeared. Antinuclear antibodies and antibodiesagainst extractable nuclear antigens were repeatedly negative.Anti-DNA-antibodies were found slightly positive.

After September 1988, she was hospitalized several timesfor prolonged vomiting, fatigue, fever, dizziness andprogressive walking difficulties with ataxia and short gait. Inaddition, impairment of memory, taste, and hearingoccurred. In February 1989, the erythrocyte sedimentationrate (ESR) was 125 mm h~', serum C-reactive protein 83 mgI"1 (normal <10 mg I"1), and leucocytes 9.2X109 I"1 with96% granulocytes. Sinus X-ray showed sinusitis, and thebrain CT showed an empty sella. Despite treatment withmethylprednisolone and i.v. erythromycin, the ESR and C-reactive protein remained elevated. At CSF examinations(February 1989 and January 1991), leucocyte counts andprotein concentrations were normal, as was the IgG/albuminratio, but one or two subfractions were observed withprotein electrophoresis. In January 1991, MRI of thebrain showed enlarged ventricles, cortical atrophy, andmarked degenerative changes in the peri ventricular areas(Fig. 1A). Total serum immunoglobulins were normal, butimmune electrophoresis showed an M-component (IgGlambda). Circulating immune complexes also occurred.Rheumatoid factor, antinuclear antibodies, anticardiolipin,TPHA (Treponema pallidum haemagglutination test), anti-phospholipid antibodies, and antibodies against 6. burgdorfehwere negative in the serum. Leucocytes were 3.5X109 1"'with an excess of band forms.

In August 1991, CSF examination showed no inflam-matory cells, a slightly elevated protein concentration of762 mg I"1, and no antibodies against B. burgdorferi. Cultureof CSF in BSK-II medium showed very slow growth ofspirochaetes during 3 months. Using monoclonal antibodies,immunofluorescence and PCR, the spirochaete was identifiedas B. burgdorferi s.l.

In December 1991, antimicrobial treatment withceftriaxone (2 g i.v. daily) was instituted. The patientimproved slightly, and therapy was continued after 3 weekswith oral amoxicillin (500 mg every 8 h) and oral probenecid(500 mg every 8 h). After 1 week on amoxicillin, the patientdeveloped urticaria. Oral doxycycline (100 mg every 12 h)was substituted and continued until July 1992. During thistreatment, the walking difficulties and fever episodes recurred.All cultures for fungi and common bacteria were negative.In January 1992, brain MRI showed slight progression ofthe periventricular lesions from the image obtained 1 yearearlier. In March and July 1992, subdural haemorrhages ofunknown origin were evacuated.

On August 7, 1992, plasma and bone marrow specimenswere positive for B. burgdorfeh PCR. Treatment withceftriaxone (2 g i.v. daily) was reinstituted, the patient

Fig. 1 (Patient 1). Enlarged ventricles, cortical atrophy and markeddegenerative changes are seen in periventricular areas on T2-weighted MRI of the brain after the first treatment (A). Within anarea of diffuse demyelination in subcortical white matter (B; LuxolFast Blue-Cresyl Violet stain: bar = 400 urn) there is mildperivascular inflammation (C; anti-CD45 immunostain + H and E

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reacting with high fever. Empirical antifungal therapy withamphotericin B was also started. These treatments werecontinued until the patient died on September 12, 1992.

At autopsy, the pathological changes were slighter thanexpected. The spleen was slightly enlarged. Chronic liverstasis and mild pulmonary oedema were detected. No signsof fungal infection were seen. Neuropathological examinationshowed a chronic left-sided subdural haematoma. Its structurewas compatible with the haemorrhages occurring 6 and 2months before death. An increased number of plasma cellswere present within the organizing connective tissue of thehaematoma. In subcortical and periventricular white matter,diffuse demyelination with mild perivascular inflammationwas seen (Fig. IB and C). In one of the six analysed braintissue specimens, B. burgdorferi DNA was detected bythe PCR.

Patient 2This 40-year-old man had previously been healthy, apartfrom reactivation of a genital herpes infection some weeksbefore. He recalled no tick bites or erythema migrans. OnDecember 26, 1992, he had a generalized seizure and wasadmitted to the hospital. Another seizure occurred on the dayof admission. Brain CT was normal. On admission, CSFexamination showed an unremarkable increase of proteinlevel (688 mg I"1) with no inflammatory cells. The PCRassays for herpes simplex virus (HSV) and antibodies againstviruses were negative in the CSF. Serum IgM antibodiesagainst B. burgdorferi were found at a low level and IgGantibodies against Chlamydia pneumoniae were moderatelyelevated. Serum C-reactive protein was 50 mg 1"', lacticdehydrogenase 927 U I"1 (normal value < 440 U I"1) andbilirubin 36 |J.mol 1~' (normal value <20 |imol l"'l), but thechanges were transient. Other laboratory tests were normalincluding serum hepatitis B surface antigen, antibodies againstHIV and herpes viruses. The EEG showed an irritative focusin the left hemisphere.

On December 30, MRI of the brain showed three smallfrontal lesions at the bottom of the left frontal lobe near themeninges. The imaging of these lesions was enhanced usingcontrast medium (Fig. 3A). In the right pleural cavity, a chestX-ray examination showed fluid, which disappeared in 2weeks. The CT showed a central cystic lesion in the leftkidney, but no abnormal findings were obtained in themediastinum, lungs, or pleural cavities. On December 31, aCSF examination showed 4X106 1"' lymphocytes, but protein(373 mg I"1), and angiotensin convertase enzyme andlysozyme concentrations were normal. The CSF antibodiesagainst herpesviruses, B. burgdorferi, and Treponemapallidum were negative as was antigen detection for HSVand PCR for B. burgdorferi. The PCR for HSV was positivewith this specimen. Culture for viruses and mycobacteriaremained negative.

On January 8, 1993, a frontally located brain lesion wasresected for suspected malignancy. Histopathological studies

p. . :

Fig. 2 (Patient 2). Gadolinium enhanced Tj-weighted MRI of thebrain (December 1992) shows three small lesions at the bottomof the left frontal lobe (A). In the surgical sample of the lesionindicated with an arrowhead in A the wall of a leptomeningealvessel is infiltrated by abundant lymphocytes (B; H and E;bar = 200 urn).

showed lymphocytes in the walls of leptomeningeal andsmall penetrating arteries as well as in the perivascularspace of the latter (Fig. 2B). The adjacent cortex wasslightly oedematous with very mild astrocytic gliosis. APCR analysis of three separate brain specimens detectedDNA of B. burgdorferi. The IgM (but not IgG) antibodiesagainst B. burgdorferi were positive only in the first pretreat-ment serum sample but negative thereafter. The circulatingimmune complexes and complement activation products werepositive. The IgG antibodies against C. pneumoniae wereelevated at a constant level, but IgM antibodies remainednegative, indicating that the IgG antibodies were of earlierorigin. A neuropsychological investigation showed memoryimpairment affecting verbal function and slightly impairedfluency of verbal expression. Anticonvulsive therapy withcarpamazepine was started.

Table 2 shows changes in the antimicrobial treatmentschedule and the development of the brain lesions appearingon MRI. During the antibiotic treatment, MRI of the brainshowed new lesions: one enhancing lesion (2 cm in diameter),suggestive of focal vasculitis, located medially from the.postoperative area, and later, enhancing lesions at the bottomof the right frontal lobe and a frontal lobe sulcus (Fig. 3Aand B). However, the initial lesion at the bottom of the leftfrontal lobe behind the orbita was now markedly smallerthan at previous examinations. Later images showed that the

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Table 2 Development of brain lesions, PCR results for B. burgdorferi, and antimicrobial therapy given to Patient 2

MRI findings PCR for B. burgdorferi Therapy (duration)

Dec. 1992

Jan. 1993

Feb. 1993

March 1993

A close group of threefrontal lesions (Fig. 2).Operative resection.

A new enhancing lesionmedially from thepostoperative area.

Three new lesionsdistant from theprimary lesions (Fig. 3).

Negative in serumand CSF.Positive in threebrain tissuespecimens.

Negative in CSF.

Acyclovir (10 days)

Ceftriaxone 2 g daily i.v. (21 days)then amoxicillin 500 mg t.i.d. +probenecid 500 mg t.i.d. p.o.(20 days).Ceftriaxone 2 g daily i.v. (28 days)+ azithromycin 250 mg daily p.o(21 days) +rifampin 600 mg dailyp.o (21 days).Cefixime 200 mg t.i.d. p.o. +probenecid 500 mg t.i.d. p.o.(100 days).

April 1993 No new lesions, thefirst foci constantlyreducing in size.

July 1993Dec. 1993

Jan. 1994March 1994April 1994

May 1994June 1994

Sept. 1994Oct. 1994

May 1995

Only postoperative changes.A new focus adjacent to thethird ventricle (Fig. 3)

The focus seen on previousMRI disappeared.A new focus in a frontalsulcus and a large peri-ventricular lesion (Fig. 3).

All lesions (including theperiventricular ones) haddisappeared.No new lesions.

Negative in CSF.

Positive in plasma.Negative in plasmaand bone marrow

Negative in plasma.

Negative in plasma.

End of antibiotic therapy.Doxycycline 150 mg t.i.d. p.o.(120 days).

End of antibiotic therapy.

Ceftriaxone 2 g daily i.v.(100 days).

End of antibiotic therapy.

first lesion was constantly reducing in size, and five monthsafter onset of antibiotic therapy all the new foci of the putativevasculitic process had also disappeared. The antibiotic therapywas discontinued on July 5, 1993.

The patient was asymptomatic at the end of therapy. Wholebody bone scanning was carried out in June 1993 becauseof a history of pain in the thoracic spine some months earlier.Slightly increased uptake of isotope in the thoracic spine wasseen, but the finding was considered unspecific. The EEGafter sleep deprivation was normal in July 1993.

Five months after the end of antibiotic therapy, brain MRIshowed a new focus located adjacent to the third ventricle(Fig. 3A and B). Oral antibiotic treatment was started (thepatient was asymptomatic; jeeTable 2). The next MRI showedthat the treatment had probably had a beneficial effect on theformer lesions, but again, a new focus in a frontal sulcusand a relatively large pathological area in periventricularwhite matter were detected (Fig. 3B). On May 17, 1994,DNA of B. burgdorferi was detected by PCR in the patient'splasma specimen (Table 2). Intravenous antibiotic therapywas reinstituted and continued for 100 days. Thereafter, on

MRI studies of the brain, all lesions and periventricularenhancement have disappeared, and no new lesions havedeveloped to date (Table 2). The antiepileptic therapy hasbeen discontinued, and no new seizures have occurred.

Patient 3In the summer of 1993, this previously healthy 11-year-oldgirl had visited an area in Southern Finland where Lymeborreliosis is endemic. In September 1993, occasionalepisodes of hyperactivity followed by headache wereobserved by her family. On October 1, she developed paresisof the right lower limb. On October 7, she was admitted toa local hospital and 1 week later to the Oulu UniversityCentral Hospital. Standing on the right leg alone was difficult,and walking was slightly impaired.

On October 13, CT of the brain showed a periventricularlow density enhancing lesion, 10X6 mm2 in diameter, andlocated in left parietal lobe white matter. The lesion wassuggestive of a neoplasm. On the next day, using MRI, thedimensions of the enhancing lesion were found to be

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B

Fig. 3 (Patient 2). New enhancing lesions during antibiotictherapy (March 1993, see Table 3.), marked with T in frontalschematic view summarized from three close MR images (A).The initial lesions (not shown) were now markedly smaller thanin previous images. In July 1993, at the end of antibiotictreatment, the MRI showed only postoperative changes in the leftfrontal lobe while all other lesions were no longer detectable. InDecember 1993 the MRI showed a new focus (marked with '2')adjacent to the third ventricle (A and B). Again, the next MRI inApril 1994 showed a new focus (marked with '3') in the frontalsulcus and a large lesion (marked with '3') in periventricularwhite matter (B). In October 1994 the MRI demonstrated thedisappearance of all lesions after vigorous antibiotic treatment(see Table 2).

40X20x8 mm3 and the surrounding oedematous area was20-30 mm thick (Fig. 4A). The EMG was normal. Abdominalultrasonography showed mild splenomegaly.

On October 22, a craniotomy was carried out. In the areaof the enhancing lesion, shown by MRI, elastic and stretchytissue with abnormal white colour was detected. Onhistological examination, focal necrotic areas were found,surrounded by foamy macrophages, reactive astrocytes andoedema. (Fig. 4B). An increased number of small vesselswith thickened walls and prominent endothelial cells werealso seen. Lymphocytes occurred in the walls of some vessels.

Fig. 4 (Patient 3). A periventricular, strongly enhancing lesionwith surrounding oedema suggestive of a neoplasm and located inleft parietal lobe white matter on Tpweighted MRI (A). A bloodvessel and its perivascular space within oedematous necrotizedbrain parenchyme is infiltrated by lymphocytes (B; H and E,bar = 50 u.m).

Haemoglobin, ESR, and serum C-reactive protein valueswere normal. Serum total immunoglobulins were normal,except for a slightly increased value of IgM at 1.94 g 1"'(normal value 0.35-1.63 g I"1). Serum rheumatoid factor,antinuclear antibodies, extractable nuclear antigens, anti-DNA- and anti-phospholipid antibodies were negative, andso were antibodies against several viruses. On November 4,lumbar puncture was carried out. The CSF specimen gave anegative virus culture and HSV PCR but B. burgdorferi PCRwas positive with two separate CSF specimens (detected attwo separate runs).

December 21, 1993, antibiotic therapy with ceftriaxone(2 g i.v. daily) was started for 4 weeks followed by therapywith oral amoxicillin (500 mg every 8 h) combined with oralprobenecid (500 mg every 8 h). On February 1, the antibiotictherapy was stopped because of bloody diarrhoea. Cultureand toxin detection for Clostridium difficile were negative.The diarrhoea was cured with oral metronidazole.

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On February 1, 1994, MRI of the brain showed reductionof abnormal tissue around the operative area. At this time,no enhancement was seen in the walls of the cavity. At afollow-up of 1 year, recovery was observed with only a slightabnormality in walking. No new symptoms had developed.

DiscussionIn this study, the evidence for the presence of B. burgdorferiwas mainly based on positive PCR results. However, in onepatient the spirochaete was also cultured from the CSF. ThePCR detected DNA of B. burgdorferi directly in the brainlesions of two patients, and in the CSF, plasma or bonemarrow of all three patients. In two patients, a positive PCRresult was obtained from more than one type of specimen.Based on the results of control specimens and on the rigorouscontamination control we consider our PCR results reliable.Since two of the patients had no previous antibiotic treatmentbefore the first PCR-positive samples were obtained, it ishighly probable that the PCR results indicate an ongoinginfection. The CSF sample obtained from the third patientwith a history of several previous antibiotic courses for otherreasons was also culture-positive. The causative role ofborrelial infection was supported by constriction or disappear-ance of the lesions in two patients after antimicrobial therapydirected against B. burgdorferi.

Both clinical and experimental studies have shown thatB. burgdorferi can rapidly and effectively penetrate the CNS(Garcia-Monco et al, 1990; Luft et al., 1992). Interactionwith the intraluminal endomelial surface by the spirochaete,directly or via systemic cytokines, was necessary for theincrease of the permeability of the blood-brain barrier (BBB).The dissemination of B. burgdorferi to various organs dependson its ability to adhere to and penetrate the endothelium, andthe BBB as well (Garcia-Monco et al., 1990). In an in vitromodel, B. burgdorferi was shown to adhere to the endothelialsurface or to an exposed subendothelial basement membraneand migrate between endothelial cells, either at their junctionsor in an area with endothelial damage (Comstock andThomas, 1989; Szczepanski et al., 1990). Recent findingsindicate that B. burgdorferi can acquire proteolytically activehost components. This mechanism could facilitate thedissemination and localization of spirochaetes to sites ofvascular injury (Klempner et al., 1995). The affinity of theorganism for astrocytes, the nearest neighbours of braincapillaries, could facilitate its access to the nervous system(Benach and Garcia-Monco, 1992). The breakdown of theBBB noted after i.v. inoculation of B. burgdorferi (Garcia-Monco et al., 1990) may be due to either generalized orfocal production of inflammation mediators after adhering ofspirochaetes to the endothelium. Focal vasculitis may thenbe developed by activation of endothelial cells and furtherrelease of inflammation mediators. Involvement of the CNSis associated with scattered peri vascular mononuclear cellinfiltrates in the cerebral cortex, mainly consisting of T-helpercells (Meurers et al., 1990). The infiltrates are sometimes

accompanied by mild, spongiform changes, a focal increasein microglial cells, and a modest infiltration of lymphocytesand plasma cells in the leptomeninges (Duray, 1989a).

Our observations support results from both experi-mental and clinical investigations suggesting that thenumber of B. burgdorferi spirochaetes in the CNS is verylow. The spirochaete could be cultivated from the CSF ofonly one of our patients. To date, almost all attempts toisolate B. burgdorferi from human brain tissue have failed.Culture of B. burgdorferi-Yike organisms from brain tissueaffected by Alzheimer's disease has been reported twice(MacDonald and Miranda, 1987; Miklossy et al., 1994). Afew other reports have been published showing the agent inbiopsy or autopsy specimens. In one patient with subacuteencephalitis, a brain biopsy specimen showed spirochaetesmorphologically compatible with B. burgdorferi and micro-gliosis without inflammatory infiltrate (Pachner et al.. 1989).In a newborn whose mother had suffered from Lyme borre-liosis in early pregnancy and who died during the first dayafter birth, B. burgdorferi could be demonstrated by stainingand immunocytochemistry in the brain and liver (Weberet al., 1988). One child with a history of severe arthritis forseveral months died during a protracted seizure which washer first neurological manifestation of the disease; histo-logical studies of brain tissue showed general vasculitis, andB. burgdorferi was demonstrated by silver staining (Millneretai, 1991). Brain involvement during experimental infectionwith B. burgdorferi has been reported in several animalmodels (Johnson et al., 1984; Burgdorfer and Gage, 1987;Barthold et al., 1988; Garcia-Monco et al., 1990; Moodyetai, \990b; Pachner and Itano, 1990; Barthold et al. 1992),but even in these experiments the presence of borreliae inthe brain or CSF has often been short-lived.

The pathogenesis of CNS manifestations in Lymeborreliosis is inadequately known. Our observations supportearlier reports suggesting that vasculitis may be one of theprimary pathophysiological mechanisms in neuroborreliosis(Camponovo and Meier, 1986; Midgard and Hofstad, 1987;Kohler et al., 1988; Meier and Grehl, 1988; Mokry et al,1990; Meurers et al., 1990). This is only logical, because B.burgdorferi infection frequently causes perivascularinflammation or vasculitis (e.g. retinal vasculitis) in affectedorgans other than the CNS (Duray. 1989a, b; Olson andEsterly, 1990; Smith et al, 1991; Karma et al, 1995). Ingeneral, micro-organisms causing retinal vasculitis have beenfound to be much the same as those causing cerebralvasculitides (Somer and Finegold, 1995). In the peripheralnervous system, inflammatory vascular changes or vasculitisseem to cause axonal degeneration in Lyme borreliosis(Camponovo and Meier, 1986; Meurers et al, 1990). Studieson experimental borrelia infections have also shownprominent lymphoplasmacellular infiltration in themicrovasculature, endarteritis obliterans, and spirochaetes inand around blood vessels of synovial and myocardial tissues(Johnston et al, 1985; Moody et al, 1990a). Vasculitis alsois a predominant finding in T. pallidum infection, another

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spirochetal disease with CNS invasion (Kohler et al., 1988;Coyle and Dattwyler, 1990). Syphilitic endarteritis may causemultiple small infarctions in the CNS, or involve the vasavasorum of large or medium-sized vessels, and lead toaneurysms or ischaemic infarction months or years afteronset of infection.

Our study supports a sporadic causative role for B.burgdorferi infection in stroke-like diseases or vasculitis inagreement with previous studies (Midgard and Hofstad, 1987;Uldry et al., 1987; Veenendaal-Hillbers et al., 1988; Mayand Jabbari, 1990; Millner et al., 1991; Hammers-Berggrenet al., 1993). This is also in agreement with the recent findingthat patients with chronic Lyme disease encephalopathyhave multifocally reduced blood perfusion to the cerebralhemispheres, particularly in white matter, and that thesepatients show objective improvement in brain perfusion afterantibiotic treatment (Steere et al, 1994).

The brain autopsy specimens of Patient 1 showed extensivedemyelination in periventricular areas, and B. burgdorferi wascultivated from the CSF. This finding, combined with the longhistory of her disease (decades), indicates that B. burgdorferimay occasionally cause pronounced demyelination, possiblyvia damage to microvasculature, similar to that seen inneurosyphilis. Several studies have found an associationbetween demyelinating disease and B. burgdorferi infection(Reik et al., 1985; Kohler et al., 1988; Pachner et al.,1989; Clavelou et al., 1993), although in multiple sclerosisB. burgdorferi infection rarely seems to be the trigger (Coyle,1989; Baig et al., 1991; Coyle et al. 1993). Direct damageto oligodendroglial cells may cause demyelination becauseB. burgdorferi very actively binds to them (Baig et al., 1991).This binding may partly explain the long-term persistence ofB. burgdorferi in the CNS and explain why the spirochaeteis seldom isolated from the CSF (Garcia-Monco et al.,1989; Pachner and Delaney, 1993). Galactocerebroside, acomponent of myelin, and other glycosphingolipids arepossible binding structures for B. burgdorferi and otherrelated spirochaetes causing disease in the central andperipheral nervous systems (Garcia-Monco et al., 1992;Backenson et al., 1995). Autoimmune reactions, triggeredby B. burgdorferi infection, may also cause demyelination.B-cells capable of responding to myelin basic protein havebeen a common finding in the CSF of patients with LNB(Baig et al., 1991). Because nervous tissue is considered animmunologically privileged site, autoreactive B-cells, andautoantibodies as their products, may arise as a result of hostresponse to neuronal antigens exposed by tissue damage.Another explanation to autoreactivity could be molecularmimicry and cross-reactions between neuronal autoantigensand antigens of B. burgdorferi (Sigal and Tatum, 1988; Sigal,1993). Experimental studies on SCID (severe combinedimmuno-deficient) mice, however, show that at leastarthritogenesis is not necessarily dependent on an intactimmune system, since chronic arthritis is inducible in thesemice (Barthold et al., 1992).

Brain lesions in Patient 2 developed in previously intact

areas during or after treatment, the last one of them 16months after onset of prolonged antibiotic therapy. Severalreports have been published on the occurrence of new fociand paradoxical enlargement of CNS lesions during thetreatment of mycobacterial CNS infections (Afghani andLieberman, 1994). The direct effects of mycobacterialproducts or the host's immunological reactions elicited bymicrobial components have been offered as the most likelyexplanation for the appearance of new foci in myco-bacterial infections (Afghani and Lieberman, 1994). Similarmechanisms could explain the development of new lesionsduring therapy in Patient 2. However, DNA of B. burgdorferiin the plasma of the patient over 16 months after the onsetof first antibiotic treatment suggests the presence of ongoinginfection. The route of entry to the new sites could havebeen the vascular wall after occurrence of subclinical spiro-chaetemia. Another explanation for the onset of new lesionsis that the spirochaetes were already at the site of lesionsbefore the antibiotic treatment. Because of metabolic inactiv-ity, they were not affected by the antibiotics. After a latentperiod, the uneradicated spirochaetes could have activatedand caused changes visible on brain MRI. The disappearenceof all lesions after repeated therapy further supports thetheory that the lesions were directly related to B. burgdorferiinfection. Our experience with this patient suggests that, inrare cases, extended or repeated antibiotic treatments may benecessary to eradicate the spirochaete from sites where it hasaquired a latent state.

The diagnosis of LNB has usually been based onnonspecific findings, serological testing and other indirectmethods. Stereotactic biopsy or a surgical operation arenecessary for direct demonstration of an etiological agent invasculitis or brain lesions. However, evidence for the presenceof B. burgdorferi could be obtained by culture or PCR ofthe CSF or plasma specimens in all three of our patients.This is consistent with the earlier finding that small numbersof spirochaetes or their structures occasionally circulate inthe blood not only in early infection but also during the laterstages of Lyme borreliosis (Viljanen et al., 1992; Nadelmanet al., 1994; Oksi et al., 1994 and 1995a, b; Oksi, 1995).Thus, MRI findings compatible with vasculitis associatedwith a positive PCR result from the CSF or plasma, evenwithout direct demonstration of the spirochaete in the brainlesions, might be an indication for antimicrobial treatmentdirected against B. burgdorferi.

Our patients had no borrelial antibodies in their CSF. Thisresult is in contrast with most published studies on Europeanneuroborreliosis patients, in whom intrathecal antibodyproduction has usually been detected. Indeed, intrathecalantibody production has been one of the criteria for thediagnosis of neuroborreliosis (Steere et al. 1990; Baig el al.,1991; Hansen and Lebech, 1991, 1992). By contrast,American patients with LNB often show no intrathecallyproduced antibody (Steere et al., 1990; Kuntzer et al., 1991;Luft et al., 1992). It is not known whether the differencesreported between American and European LNB patients are

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due to patient selection or some other factor (Steere et al,1990). However, our results indicate that the differencesbetween European LNB and its North-American counterpartmay not be as great as has been suggested. Three plausibleexplanations for the lack of intrathecal antibody productionin LNB may be suggested. First, the CNS can be consideredan immunoprivileged site where the spirochaete can lie latentout of reach of the host immune system (Guy and Turner,1989; Pfister et al, 1989; Halperin et al., 1991). Second,studies with T cell clones suggest that B. burgdorferi mayshift the immune response of the host towards cell-mediatedimmunity at the expense of antibody production (Ysselet al., 1991). This is advantageous from the spirochaete'spoint of view, because antibodies are obviously the majorfactor in the host defense against B. burgdorferi infection(Fikrig et al., 1992). We have also obtained evidence thatB. burgdorferi infection can cause suppression of Th2 cellsand activation of Thl cells in patients with late Lymeborreliosis (Oksi et al., 1996). Third, immune complexformation and binding of antibodies to complexes may be amechanism underlying the negative results of routine antibodyassays (Schutzer et al., 1990).

Hemiparesis (Uldry et al., 1987; Veenendaal-Hilbers et al,1988; May and Jabbari, 1990), which was the main manifesta-tion in one of our patients, and epilepsy (Millner et al, 1991;Mourin et al, 1993), the dominating symptom of another"patient, have also been reported to be associated with LNB.The almost complete recovery of our patient from thehemiparesis, and the complete disappearance of brain fociand recovery from the epilepsy in another patient afterantimicrobial treatment may suggest infectious etiology. Werecommend exclusion of Lyme borreliosis as a trigger ofdisease in selected patients with demyelinating disease,hemiparesis, or epilepsy.

We conclude that cerebral lymphocytic vasculitis andmultifocal encephalitis may be associated with B. burgdorferiinfection. The presence of B. burgdorferi DNA in tissuesamples from areas with inflammatory changes indicates thatdirect invasion of B. burgdorferi may be the pathogeneticmechanism for focal encephalitis in Lyme neuroborreliosis.

AcknowledgementsThe language of this manuscript was revised by Simo Merne.This study was financially supported by the Emil AaltonenFoundation, the Maud Kuistila Foundation, the OrionCorporation Research Foundation, the Turku UniversityFoundation and the Finnish Medical Foundation. Parts ofthis paper were presented at the VI International Conferenceon Lyme borreliosis, Bologna, Italy, June 19-22, 1994, andat the Symposium on the Therapy and Prophylaxis for Lymeborreliosis, Portoroz, Slovenia, May 13-16, 1995.

ReferencesAfghani B, Lieberman JM. Paradoxical enlargement or developmentof intracranial tuberculomas during therapy: case report and review.[Review]. Clin Infect Dis 1994; 19: 1092-9.

Backenson PB, Coleman JL, Benach JL. Borrelia burgdorferi showsspecificity of binding to glycosphingolipids. Infect Immun 1995;63: 2811-7.

Baig S, Olsson T, Hojeberg B, Link H. Cells secreting antibodiesto myelin basic protein in cerebrospinal fluid of patients with Lymeneuroborreliosis. Neurology 1991; 41: 581-7.

Barthold SW, Moody KD, Terwilliger GA, Duray PH, Jacoby RO,Steere AC. Experimental Lyme arthritis in rats infected with Borreliaburgdorferi. J Infect Dis 1988; 157: 842-6.

Barthold SW, Sidman CL, Smith AL. Lyme borreliosis in geneticallyresistant and susceptible mice with severe combinedimmunodeficiency. Am J Trop Med Hyg 1992; 47: 605-13.

Belman AL, Coyle PK, Roque C, Cantos E. MRI findings inchildren infected by Borrelia burgdorferi. Pediatr Neurol 1992; 8:428-31.

Benach JL, Garcia Monco JC. Aspects of the pathogenesis ofneuroborreliosis. In: Schutzer SE, editor. Lyme disease: molecularand immunologic approaches. New York: Cold Spring HarborLaboratory Press, 1992: 1-10.

Burgdorfer W, Gage KL. Susceptibility of the hispid cotton rat(Sigmodon hispidus) to the Lyme disease spirochete (Borreliaburgdorferi). Am J Trop Med Hyg 1987; 37: 624-8.

Camponovo F, Meier C. Neuropathy of vasculitic origin in a caseof Garin-Boujadoux-Bannwarth syndrome with positive borreliaantibody response. J Neurol 1986; 233: 69-72.

Clavelou P, Vernay D, Cuoq N, Soubrier M, D'Hombres A,Dordain G, Toumilhac M. Demyelinating involvement in Bonrelianneuropathies. [French]. Rev Neurol (Paris) 1993; 149: 320-5.

Comstock LE, Thomas DD. Penetration of endothelial cellmonolayers by Borrelia burgdorferi. Infect Immun 1989; 57:1626-8.

Coyle PK. Borrelia burgdorferi antibodies in multiple sclerosispatients. Neurology 1989; 39' 760-1.

Coyle PK, Dattwyler R. Spirochetal infection of the central nervoussystem. [Review]. Infect Dis Clin North Am 1990; 4: 731^16

Coyle PK, Krupp LB, Doscher C. Significance of reactive Lymeserology in multiple sclerosis. Ann Neurol 1993; 34: 745—7.

Duray PH. Clinical pathologic correlations of Lyme disease. RevInfect Dis 1989a; 11 Suppl 6: S1487-93.

Duray PH. Histopathology of clinical phases of human Lymedisease. [Review]. Rheum Dis Clin North Am 1989b; 15: 691-710.

Fikrig E, Barthold SW, Marcantonio N, Deponte K, Kantor FS,Flavell RA. Roles of OspA, OspB, and flagellin in protectiveimmunity to Lyme borreliosis in laboratory mice. Infect Immun1992; 60: 657-61.

Garcia-Monco JC, Benach JL. Lyme neuroborreliosis. [Review].Ann Neurol 1995; 37: 691-702.

Garcia-Monco JC, Fernandez-Villar B, Benach JL. Adherence ofthe Lyme disease spirochete to glial cells and cells of glial origin.J Infect Dis 1989; 160: 497-506.

Garcia-Monco JC, Villar BF, Alen JC. Benach JL. Borrelia

by guest on Septem

ber 14, 2010brain.oxfordjournals.org

Dow

nloaded from

CNS findings in Lyme borreliosis 2153

burgdorferi in the central nervous system: experimental and clinicalevidence for early invasion. J Infect Dis 1990; 161: 1187-93.

Garcia-Monco JC, Fernandez-Villar B, Rogers RC, Szczepanski A,Wheeler CM, Benach JL. Borrelia burgdorferi and other relatedspirochetes bind to galactocerebroside. Neurology 1992; 42: 1341-8.

Guy EC, Turner AM. Seronegative neuroborreliosis [letter]. Lancet1989; 1: 441.

Halperin JJ, Volkman DJ, Wu P. Central nervous systemabnormalities in Lyme neuroborreliosis. Neurology 1991: 41:1571-82.

Hammers-Berggren S, Grondahl A, Karlsson M, von Arbin M, ,Carlsson A, Stiernstedt G. Screening for neuroborreliosis in patientswith stroke. Stroke 1993; 24: 1393-6.

Hance AJ, Grandchamp B, LeVy-Frebault V, Lecossier D, RauzierJ, Bocart D, et al. Detection and identification of mycobactena byamplification of mycobacterial DNA. Mol Microbiol 1989; 3: 843-9.

Hansen K, Lebech AM. Lyme neuroborreliosis: a new sensitivediagnostic assay for intrathecal synthesis of Borrelia burgdorferi—specific immunoglobulin G, A, and M. Ann Neurol 1991; 30:197-205.

Hansen K, Lebech AM. The clinical and epidemiological profile ofLyme neuroborreliosis in Denmark 1985—1990. A prospective studyof 187 patients with Borrelia burgdorferi specific intrathecalantibody production. Brain 1992; 115: 399-423.

He Q, Marjamaki M, Soini H, Mertsola J, Viljanen MK. Primersare decisive for sensitivity of PCR. Biotechniques 1994; 17: 82-7.

Johnson RC, Marek N, Kodner C. Infection of Syrian hamsterswith Lyme disease spirochetes. JClin Microbiol 1984; 20: 1099-101.

Johnston YE, Duray PH, Steere AC, Kashganan M, Buza J,Malawista SE, et al. Lyme arthritis. Spirochetes found in synovialmicroangiopathic lesions. Am J Pathol 1985; 118: 26-34.

Karma A, Seppala I, Mikkiia H, Kaakkola S, Viljanen M, TarkkanenA. Diagnosis and clinical characteristics of ocular Lyme borreliosis.Am J Ophthalmol 1995; 119: 127-35.

Klempner MS, Noring R, Epstein MP, McCloud B, Hu R, LimentaniSA, et al. Binding of human plasminogen and urokinase-typeplasminogen activator to the Lyme disease spirochete, Borreliaburgdorferi. J Infect Dis 1995; 171: 1258-65.

Kohler J, Kern U, Kasper J, Rhese-Kupper B, Thoden U. Chroniccentral nervous system involvement in Lyme borreliosis. Neurology1988; 38: 863-7.

KrUger WH, Pulz M. Detection of Borrelia burgdorferi incerebrospinal fluid by the polymerase chain reaction. J MedMicrobiol 1991; 35: 98-102.

Kuntzer T, Bogousslavsky J, Miklossy J, Steck AJ, Janzer R, RegliF. Borrelia rhombencephalomyelopathy. Arch Neurol 1991; 48:832-6.

Luft BJ, Steinman CR, Neimark HC, Muralidhar B, Rush T, FinkelMF, et al. Invasion of the central nervous system by Borreliaburgdorferi in acute disseminated infection [published erratumappears in JAMA 1992; 268: 872] [see comments]. JAMA 1992;267: 1364-7. Comment in: JAMA 1992; 268: 872.

MacDonald AB, Miranda JM. Concurrent neocortical borreliosisand Alzheimer's disease. Hum Pathol 1987; 18: 759-61.

May EF, Jabbari B. Stroke in neuroborreliosis. [Review]. Stroke1990; 21: 1232-5.

Meier C, Grehl H. Vasculitic neuropathy in the Garin-Bujadoux-Bannwarth syndrome. A contribution to the understanding of thepathology and pathogenesis of the neurological complications inLyme borreliosis. [German] Dtsch Med Wochenschr 1988; 113:135-8.

Meurers B, Kohlhepp W, Gold R, Rohrbach E, Mertens HG.Histopathological findings in the central and peripheral nervoussystems in neuroborreliosis. A report of three cases. J Neurol 1990;237: 113-6.

Midgard R, Hofstad H. Unusual manifestations of nervous systemBorrelia burgdorferi infection. Arch Neurol 1987; 44: 781-3.

Miklossy J, Kasas S, Janzer RC, Ardizzoni F, Van der Loos H.Further ultrastructural evidence that spirochaetes may play a rolein the aetiology of Alzheimer's disease. Neuroreport 1994; 5:1201^.

Millner MM, Mullegger RR, Spork KD, Stanek G. Lyme borreliosisof central nervous system (CNS) in children: a diagnostic challenge.Infection 1991; 19: 273-8.

Mokry M, Flaschka G, Kleinert G, Kleinert R, Fazekas F, Kopp W.Chronic Lyme disease with an expansive gTanulomatous lesion inthe cerebellopontine angle. Neurosurgery 1990; 27: 446-51.

Moody KD, Barthold SW, Terwilliger GA, Beck DS, Hansen GM,Jacoby RO. Experimental chronic Lyme borreliosis in Lewis rats.Am J Trop Med Hyg 1990a; 42: 165-74.

Moody KD, Barthold SW, Terwilliger GA. Lyme borreliosis inlaboratory animals: effect of host species and in vitro passage ofBorrelia burgdorferi. Am J Trop Med Hyg 1990b; 43: 87-92.

Mourin S, Bonnier C, Bigaignon G, Lyon G. Epilepsy disclosingneuroborreliosis. [French]. Rev Neurol (Paris) 1993; 149: 489-91.

Nadelman RB, Schwartz I, Wormser GP. Detecting Borreliaburgdorferi in blood from patients with Lyme disease [letter;comment]. J Infect Dis 1994; 169: 1410-1. Comment on: J InfectDis 1993; 168: 1541-3.

Oksi J. Lyme borreliosis [letter; comment]. Lancet 1995; 345: 1437.Comment on: Lancet 1995; 345: 842^1.

Oksi J, Mertsola J, Reunanen M, Marjamaki M, Viljanen MK.Subacute multiple-site osteomyelitis caused by Borrelia burgdorferi.Clin Infect Dis 1994; 19: 891-6.

Oksi J, Uksila J, Marjamaki M, Nikoskelainen J, Viljanen MK.Antibodies against whole sonicated Borrelia burgdorferi spirochetes,41-kilodalton flagellin, and P39 protein in patients with PCR- orculture-proven late Lyme borreliosis. J Clin Microbiol 1995a; 33:226CM.

Oksi J, Marjamaki M, Koski K, Nikoskelainen J, Viljanen MK.Bilateral facial palsy and meningitis caused by Borrelia doubleinfection [letter]. Lancet 1995b; 345: 1583—4.

Oksi J, Savolainen J, Pene J, Bosquet J, Laippala P, Viljanen MK.Decreased interIeukin-4 and increased gamma interferon production

by guest on Septem

ber 14, 2010brain.oxfordjournals.org

Dow

nloaded from

2154 J. Oksi et al.

by peripheral blood mononuclear cells of patients with Lymeborreliosis. Infect Immun 1996; 64: 3620-23.

Olson JC, Esterly NB. Urticarial vasculitis and Lyme disease [seecomments]. J Am Acad Dermatol 1990; 22: 1114-6.

Pachner AR, Delaney E. The polymerase chain reaction in thediagnosis of Lyme neuroborreliosis. Ann Neurol 1993; 34: 544-50.

Pachner AR, Itano A. Borrelia burgdorferi infection of the brain:characterization of the organism and response to antibiotics andimmune sera in the mouse model [see comments]. Neurology 1990;40: 1535-^0. Comment in: Neurology 1991; 41: 463.

Pachner AR, Duray P, Steere AC. Central nervous systemmanifestations of Lyme disease. Arch Neurol 1989; 46: 790-5.

Pfister HW, Preac-Mursic V, Wilske B, Einhaupl KM, WeinbergerK. Latent Lyme neuroborreliosis: presence of Borrelia burgdorferiin the cerebrospinal fluid without concurrent inflammatory signs.Neurology 1989; 39: 1118-20.

Picken RN. Polymerase chain reaction primers and probes derivedfrom flagellin gene sequences for specific detection of the agentsof Lyme disease and North American relapsing fever. J ClinMicrobiol 1992; 30: 99-114.

Reik L Jr, Smith L, Khan A, Nelson W. Demyelinatingencephalopathy in Lyme disease. Neurology 1985; 35: 267-9.

Schutzer SE, Coyle PK, Belman AL, Gohghtly MG, Drulle J.Sequestration of antibody to Borrelia burgdorferi in immunecomplexes in seronegative Lyme disease. Lancet 1990; 335: 312-5.

Sellati TJ, Burns MJ, Ficazzola MA, Furie MB. Borrelia burgdorferiupregulates expression of adhesion molecules on endothelial cellsand promotes transendothelial migration of neutrophils in vitro.Infect Immun 1995: 63: 4439-47.

Sigal LH. Cross-reactivity between Borrelia burgdorferi flagellinand a human axonal 64,000 molecular weight protein. J Infect Dis1993, 167: 1372-8.

Sigal LH, Tatum AH. Lyme disease patients' serum contains IgMantibodies to Borrelia burgdorferi that cross-react with neuronalantigens. Neurology 1988; 38: 1439^2.

Smith JL, Winward KE, Nicholson DF, Albert DW. Retinal vasculitisin Lyme borreliosis. J Clin Neuroophthalmol 1991; 11: 7-15.

Somer T, Finegold SM. Vasculitides associated with infections,immunization, and antimicrobial drugs. [Review]. Clin Infect Dis1995; 20: 1010-36.

Steere AC, Berardi VP, Weeks KE, Logigian EL, Ackermann R.Evaluation of the intrathecal antibody response to Borrelia

burgdorferi as a diagnostic test for Lyme neuroborrehosis. J InfectDis 1990; 161: 1203-9.

Steere AC, Kalish RA, Kaplan RF, Taylor E, Logigian EL. Clinicalmanifestations of Lyme disease: Chronic arthritis andencephalopathy. In: Cevenini R, Sambri V, La Placa M, editors.Proceedings of the VI International Conference on Lyme Borreliosis.Bologna: Societa Editrice Esculapio, 1994: 127-31.

Szczepanski A, Furie MB, Benach JL, Lane BP, Fleit HB. Interactionbetween Borrelia burgdorferi and endothelium in vitro. J Clin Invest1990; 85: 1637-^7.

Uldry PA, Regli F, Bogousslavsky J. Cerebral angiopathy andrecurrent strokes following Borrelia burgdorferi infection [letter]. JNeurol Neurosurg Psychiatry 1987; 50: 1703-4.

Veenendaal-Hilbers JA, Perquin WV, Hoogland PH, Doornbos L.Basal meningovasculitis and occlusion of the basilar artery in twocases of Borrelia burgdorferi infection. Neurology 1988; 38: 1317-9.

Viljanen MK, Punnonen J. The effect of storage of antigen-coatedpolystyrene microwells on the detection of antibodies againstBorrelia burgdorferi by enzyme immunoassay (EIA). J ImmunolMethods 1989; 124: 137-41.

Viljanen MK, Oksi J, Salomaa P, Skurnik M, Peltonen R, KalimoH. Cultivation of Borrelia burgdorferi from the blood and asubcutaneous lesion of a patient with relapsing febrile nodularnonsuppurative panniculitis [letter]. J Infect Dis 1992; 165: 596-7.

Wallich R, Moter SE, Simon MM, Ebnet K, Heiberger A, KramerMD. The Borrelia burgdorferi flagellum-associated 41-kilodaltonantigen (flagellin): molecular cloning, expression, and amplificationof the gene. Infect Immun 1990; 58: 1711-9.

Weber K, Bratzke HJ, Neubert U, Wilske B, Duray PH. Borreliaburgdorferi in a newborn despite oral penicillin for Lyme borreliosisduring pregnancy. Pediatr Infect Dis J 1988; 7: 286-9.

Weder B, Wiedersheim P, Matter L, Steck A, Otto F. Chronicprogressive neurological involvement in Borrelia burgdorferiinfection. J Neurol 1987; 234: 40-3.

Wokke JH, van Gijn J, Elderson A, Stanek G. Chronic forms ofBorrelia burgdorferi infection of the nervous system. Neurology1987; 37: 1031^.

Yssel H, Shanafelt MC, Soderberg C, Schneider PV, Anzola J, PeltzG. Borrelia burgdorferi activates a T helper type 1 -like T cell subsetin Lyme arthritis. J Exp Med 1991; 174: 593-601.

Received April 30, 1996. Revised June 4, 1996.Accepted July 25, 1996

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