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Association between Multiple Sclerosis and Cystic Structures in Cerebrospinal
Fluid
0. Brorson, S.-H. Brorson, T.-H. Henriksen, P.R. Skogen, R.Schoyen
Abstract
Background: The aim of the study was to search for infectious agents in the
cerebrospinal fluid (CSF) of patients with multiple sclerosis (MS).
Patients and Methods: CSF from ten patients with the diagnosis relapsing
remitting MS and from five controls without MS were examined by transmission
electron microscopy {1EM). dark field microscopy (OF), interference contrast
microscopy (ICM) and UV-microscopic examination of acridine orange staining
(AO). All CSF samples from patients and controls were cultured.
Results: Cystic structures were observed in CSF of all ten patients by AO and
TEM. OF revealed eight cyst-positive patients out of nine. One of five control
persons had such structures in the CSF; this person had suffered from erythema
migrans. Spirochete or rod-like structures emerged after culturing two of the
MS patient CSF samples and these structures could be propagated.
Conclusion: A significant association of CSF cysts and MS was identified in
this small study among residents in a coastal area of southern Norway. The
cysts could be of spirochetal origin. Our study may encourage other researchers
to study larger patient groups.
KeyWords Spirochetes. Multiple sclerosis. Cerebrospinal fluid. CNS. Electron
Microscopy
Infection 2001; 29: 315-319 DOl 10.1007/S15010-001-9144-Y
Introduction
Multiple Sclerosis (MS) is a serious disease attacking the nervous system, and
many hypotheses have been suggested to reveal the cause of the disease. The
etiologic agent responsible for the development of MS has not yet been
discovered, but MS has been suggested to be triggered by various viral
infections, e.g. human herpesvirus 6, rubella, measles, and by Chlamydia
pneumoniae [1-3]. Autoimmune disorders have also been proposed as the cause of
the disease [4], but no definite verification is given. From 1909 until the
1950s many researchers considered MS to have a spirochetal origin
because of its similarity to other spirochetal diseases [5]. Steiner [6]
observed Spirochaeta myelophthora in brain lesions from autopsied MS patients
in the 1950s. When Adams et al. [7] inoculated Macacus rhesus monkeys with
material from cases of disseminated sclerosis, spirochetes were observed in the
ventricular fluid after some months. MS has also been proposed to be caused by
an oral spirochete [8, 9].
As the course of the disease was apparently not altered by antibiotics and
observations of spirochetes in brains of MS patients could only be evidenced in
a few case records, this view was eventually abandoned. However, when entering
into the CNS, microorganisms can undergo extensive structural, antigenic and
metabolic changes which prevent them from being recognized by serology and
other diagnostic test methods [10]. These changes could also protect them from
the immune system and reduce the effect of antibiotics.
We recently observed that transformation of the spirochete Borrelia
burgdorferi to cystic forms occurs invariably and rapidly after incubation in
CSF and that they can reconvert to spirochetes if the conditions become
favorable [11]. Using light microscopy and transmission electron microscopy
(TEM), we identified morphologically similar cysts in the CSF of one MS patient
who had been treated for 4 months with intravenous ceftriaxone (unpublished
observations). Therefore, we performed this small controlled
study to examine if cystic structures are more commonly found in the CSF of
MS patients than in control individuals.
Patients and Methods
Patients
Ten patients were studied. Clinically, all of them had definite relapsing
remitting MS according to Poser's criteria (12]. All patients stayed in
hospital for prednisolone (parenteral) or beta-interferon (intramuscular)
treatment. They had been treated in this way for a few days before CSF puncture
and blood sampling. The age ranged from
34-53 years, with a mean of 44.4 years. Five males and five females
participated. The duration of the disease was in the range of 2-23 years, mean
10.4 years.
Controls
The control group consisted of five persons with the diagnosis of suspected
ischialgia. These persons stayed in the hospital for a definite diagnosis. The
age ranged from 38-63 years. with a mean of 52 years. Three males and two
females participated. One of these persons had had erythema migrans- a disease
caused by the spirochete B. burgdorferi. We performed all laboratory tests on
the CSF from this person to see the test results in a patient with this
spirochetal disease. However, he was excluded from the statistics.
All spinal punctures and blood collections were performed between February and
July 1998 at the Department of Neurology, Vestfold Sentralsykehus, Tonsberg,
Norway. Serum and CSF were stored in a refrigerator at 4 °C, examined within 1
week and then maintained at -70 °C.
The examinations were performed as blind studies as the observers did not know
whether the sample was from the patient group or the control group.
Ethics
All participants were told about eventual adverse effects of the spinal
puncture, and they signed a consent form in accordance with the advisory board
of the regional ethical commission.
Culturing
4 ml CSF was centrifuged at 6,00)g for 30 min and 0.1 ml was transferred to 4
ml BSK-H medium (Sigma no. B3528; Sigma, St. Louis, MO.USA). BSK-H medium
consists of several amino acids, vitamins, glucose and bovine albumin [13] and
is a medium for culturing the spirochete B. burgdorferi. The concentration of
inactivated (56 °C. 30 min) rabbit serum (Sigma R7136) or fetal calf (cat
no.14-701 E: Bio Whittaker Boehringer
lngelheim Bioproducts Partnership, Verviers, Belgium) the serum in the BSK-H
medium was 6% and was free of antibodies against B. burgdorferi. All culture
media had been sterile filtered by the manufacturing company (Sigma) ensuring
both sterility and
the absence of mammalian cells from serum. In addition, we filtered the media
with a 0.2 um filter (Schleicher & Schuell FP 030{2, Dassel, Germany) to remove
small particles which may resemble cysts in the microscope. All cultures were
incubated in sterile 5 mI closed tubes (Nalgene cryovial; Nalge, Rotherwa, UK).
BSK-H medium supplemented with rabbit serum was incubated at 30 cC and BSK-H
medium supplemented with calf serum at 22 °C. After 2 weeks of cultivation the
tubes were centrifuged at 2,770 g for 30
min. to a mixture consisting of the sediment and 0.5 ml of the supcmatant, 4 ml
of fresh medium was added. For cultures incubated at 30 °C this process was
continued in cycles of 2 weeks until an age of 3 months was reached and then
every month until 14-18 months. For the cultivation medium which was incubated
at 22 °C the same procedure was performed every 1-3 months. .
Microscopic Examination of Cultures and CSF
All sediments were examined using dark field microscopy (DF) (2(X)--EOOX) and
interference contrast microscopy (ICM) (800--2,COOx) (Zeiss Axiophot; Carl
Zeiss, Oberkochen, Germany) to determine whether any bacteria or cysts were
present.
Acridine Orange Staining (AO)
An air-dried and heat-fixed sediment of cultUred CSF (produced in the same
manner as above) was incubated with acridine orange (SO mg/l in phosphate
buffer, pH 6.4) for 4 minutes on a glass slide. Slides were subsequently
rinsed in distilled water, air-dried and examined in the UV microscope
(400-1,200x).
Transmission Electron Microscopy (TEM)
The CSF cultures were examined by TEM after 4-7 months of incubation in BSK-H
medium at 22 °C.
The examination by TEM was performed according to the following procedure. The
culture was centrifuged at 14,(XX) x 9 for 20 min. The medium was removed and
replaced with 2% glutaraldehyde in 0.2M cacodylate buffer (pH 73) and the
sediment was fixed for 2 hours. The sediment was post-fixed in 1% osmium
tetroxide in 02 M cacodylate buffer for 2 h. The pellet was dehydrated.
infiltrated and embedded in conventional epoxy resin (LX-112 ; Ladd.
Burlington, Vl~ USA) by a method described previously [14, 15]. Ultrathin
sections were cut with a diamond knife (Jumdi;Juniper ultra Micro. Stockholm.
Sweden) on an ultramicrotome (LKB 2088 UItrotom V) and mounted on 200 mesh
copper rim. The sections were stained with 5% uranyl aretate in 30% ethanol for
20 min and with Reynolds lead citrate for 5 min. The sections were examined in
a Jeol 1200 EX electron microscope to identify interesting structures.
The CSF samples were also processed for immunoelectron microscopy. The
specimens were fixed in 4 '% paraformaldehyde in phosphate buffer pH 7.3 and
the embedding was performed with LR- White using a method described previously
[16]. After blocking nonspecific labeling [17], immunogold labeling was
performed by applying a primary antibody against spirochetes (polyclonal
anti-Borrelia with known reactivity to other Borrelia (cat. no: OWYH, Dade
Behring. Marburg. Germany). dilution 1 : 2048 in 10% BSA. overnight incubation
at 4 °c. The secondary immunoreagent, antibodies coupled
to 10 nm colloidal gold particles (goat anti-rabbit IgG. Auroprobe EM Gar Gl0,
Amersham, Little Chalfont, Bucks, England) was diluted I: 50 in 3% BSA and
incubated 75 min at 22 °c. After immunolabeling. the sections were stained with
uranyl acetate and lead citrate and examined in the electron microscope as
stated above.
Fisher's exact test was applied for the statistical comparison of the
occurrence of cysts in MS patients and controls.
PCR.
PCR was performed for the CSF from all patients using standard primers for
the OspA gene of B. burgdorferi.
Analysis of IgG
Measurements of IgG in the CSF and serum were performed at the clinical
chemistry laboratory. Vestfold Sentralsykehus. Tonsberg, Norway, according to
established methods.
The IgG index was calculated automatically using the formula
IgG index= (IgG in CSF) x (albumin in serum)
_____________________________
(IgG in serum) x (albumin in CSF)
TABLE 1
Results
The main results are given in table 1. Cystic structures were observed in all
the CSF samples from the MS patients. No cysts were detected in the CSF of any
of the four controls by any method used. Cysts were detected by DF before
cu1turing in eight of the MS CSF samples (p < 0.007) and in all the MS patients
by TEM and AO (p = 0.001). AO and TEM were performed only after culturing.
Cysts were also detected with all the methods in the CSF of the patient who had
a history of erythema migrans.
Culturing
From the CSF of two MS patients, spirochete-like structures emerged after 5-7
months incubation and were visualized by DF and TEM (Figure 1). None of these
structures could be cultivated on blood or chocolate agar plates incubated at
35 °C in 5% CO2,microaerobic or anaerobic atmosphere for 5 days.
DF,ICM. and AO
By direct DF (Figure 2), ICM microscopy (after centrifugation) and AO, single
cysts (1-5 fJm) and cysts in clusters were observed, some of which had core
structures. When DF was performed before the culturing process relatively few
cysts were observed, while the
concentration of cysts was much higher after cultivation.
Figure 1. Curved spirochete-Iike bacteria (large arrow) have emerged after
cultivating CSF in BSK-H medium. Cystic forms (medium size arrows) and blebs
(small arrows) are also present. rEM. Bar = 500 nm.[Unable to display image]
Figure 2. Cysts from an MS patient examined by OF. Bar = 5 um.
TEM
TEM revealed cystic structures in the spinal fluids of all MS patients and the
patient with a history of erythema migrans (Figure 3). As seen in Figure 4, we
observed cystic structures that were intensely imrnunolabeled with a polyclonal
antispirochetal antiserum along the cell envelope. This antiserum was produced
against B. burgdorfen, but is also known to react with other spirochetes
(Treponema pallidum, Borrelia hermsii and Borrelia parkerii).
PCR
All CSFs gave a negative PCR-result with primers for the OspA gene of B.
burgdorferi. The corresponding PCR for known spirochetal forms of B.
burgdorferi was positive.
Figure 3 [Unable to display image]cysts from an MS patient observed nn the
electron microscope. The cyst is filled with biological substances and some
pycrIotic structures. TEM. Bar = 500 nm.
Discussion
Cysts were significantly associated with MS patients using each of the three
different methods. This multi-method approach strengthens the association
between CSF cysts and MS in a well-defined coastal area of southern Norway.
There are two possible explanations for the association between MS and CSF
cysts: the cystic structures are either the agents causing MS or they have
appeared in the CSF as a consequence of MS.
The positive reaction with antispirochetal antiserum, the similarity of the
cystic structures with cystic forms of spirochetes and the similarities between
the cysts in the erythema migrans patient and the MS patients suggest that the
patients were infected with a spirochete. The appearance of rod-like,slightly
curved bacteria and spirochetes after culturing two of the CSF samples in BSK-H
medium suggests the same. Spirochetes may vary in appearance and may sometimes
emerge as rod-like structures [18]. The fact that only two spinal fluids gave
rise to spirochete-like structures after culturing may be caused by the fact
that cystic forms of spirochetes may often be difficult to convert to normal
bacteria {11) and the BSK-H medium is not necessarily optimal for this possible
unknown spirochete.
[Unable to display image]
Figure 4a- A cyst from an MS patient which is immunogold labeled with
anti-Bonelia. There is a distinct and specific immunolabelng along the
envelope of the
cyst. TEM. Bar = 500 nm.
Figure 4b. Larger magnification of the envelope of the cyst to illustrate the
immunogold labeling. TEM. Bar = 200 nm.
It could be argued that the damage which MS caused in the brains of the
patients had made them more vulnerable to spirochetal infection. But this does
not seem a probable explanation. since all the MS patients had these cystic
structures in their CFS. Other researchers have proposed that spirochetes could
be the agents responsible for MS [5,
6, 8, 9]. For instance. Steiner [6] found spirochetes and granular bodies in
brain autopsies of MS patients. These were proposed to belong to the genus
BorreIia and were named Spirochaeta myelophthora [6].
We previously studied spirochetes (B. burgdorferi) that have converted from
spirochetes to cystic forms in CSF in vitro using the same methods as mentioned
above [11]. With all these methods used in this study (TEM, AO, DF), the
cystic structures observed in the CSF of the MS patients are morphologically
similar to cystic forms of spirochetes. We found that cysts which are produced
by inoculating B. burgdorferi in CSF at 37 °C can be PCR negative using
conventional DNA extraction and OspA primers (unpublished observation). This is
either because the cyst wall inhibits the entrance to the genome or because the
genomes of spirochetes have been changed. We have also to keep in mind that PCR
detection of B. burgdorferi spirochetes often may give false-negative results
[19).
The positive IgG index associated with MS in our patient cohort proves that
the patients had an active inflammatory process in the CNS (Table 1).
Inflammatory processes in the b~ and spinal cord of virtually any cause are
usually less intense than inflammation in peripheral tissues and some
microbiological agents, including spirochetes, provoke a very gentle
inflammatory response [20, 21 ]. Considering the nature of MS, this disease
could very well be a chronic infection and the clinical picture of MS has
repeatedly been confused with neuroborreliosis [22-26). Therefore, we have
both microbiological and some clinical support for the hypothesis that the
cystic structures found in the CSF of the MS patients may originate from
spirochetes which could be the causative agents of MS.
Considering the negative PCR for B. burgdorferi, the search for the etiology
of MS should continue within the family of spirochetes. This study will
encourage the efforts to identify the etiology of MS in different parts of the
world and in larger patient cohorts.
Acknowledgments
We would like to thank Rolf Komeliussen and Frank Rl}rstad, Vestfold
Sentralsykehus,Norway, for excellent technical assistance. The PCR analysis
was performed by Andrew Jenkins PhD, A/S Telelab, Skien, Norway.
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Dept. of Microbiology. Vestfold Sentralsykehus. N-3116 Tenbe
S.-H- Brorson (corresponding author) Dept. of Pathology. Ulleval University
Hospital,
N-o407 Oslo, Norway: phone: (+47/22) ,-8935, fax: -8239. e-mail:
xyz...@altavista.net
0. Brorson et al. CSF Cysts in Multiple Sclerosis