Infection 2001 Dec;29(6):315-9
PATIENTS AND METHODS: CSF from 10 patients with the diagnosis relapsing remitting MS and from five controls without MS were examined by
RESULTS:
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.
PMID: 11787831 [PubMed - in process]
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ceftriaxone (unpublished observations).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.
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.
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.
The IgG index was calculated automatically using the formula
(IgG in CSF) x (albumin in serum)
IgG index = ------------------------------------------
(IgG in serum) x (albumin in CSF)
TABLE 1
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).
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.
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.
Figure 4a [Unable to display image] 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.
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