Perturbed Microbiota/Immune Homeostasis in Multiple Sclerosis.

OBJECTIVE: Based on animal models and human studies, there is now strong suspicion that host/microbiota mutualism in the context of gut microbial dysbiosis could influence immunity and multiple sclerosis (MS) evolution. Our goal was to seek evidence of deregulated microbiota-induced systemic immune responses in patients with MS. METHODS: We investigated gut and systemic commensal-specific antibody responses in healthy controls (n = 32), patients with relapsing-remitting MS (n = 30), and individuals with clinically isolated syndromes (CISs) (n = 15). Gut microbiota composition and diversity were compared between controls and patients by analysis of 16S ribosomal ribonucleic acid (rRNA) sequencing. Autologous microbiota and cultivable bacterial strains were used in bacterial flow cytometry assays to quantify autologous serum IgG and secretory IgA responses to microbiota. IgG-bound bacteria were sorted by flow cytometry and identified using 16S rRNA sequencing. RESULTS: We show that commensal-specific gut IgA responses are drastically reduced in patients with severe MS, disease severity being correlated with the IgA-coated fecal microbiota fraction (r = -0.647, p < 0.0001). At the same time, IgA-unbound bacteria elicit qualitatively broad and quantitatively increased serum IgG responses in patients with MS and CIS compared with controls (4.1% and 2.5% vs 1.9%, respectively, p < 0.001). CONCLUSIONS: Gut and systemic microbiota/immune homeostasis are perturbed in MS. Our results argue that defective IgA responses in MS are linked to a breakdown of systemic tolerance to gut microbiota leading to an enhanced triggering of systemic IgG immunity against gut commensals occurring early in MS.

Gut Microbiome in Progressive Multiple Sclerosis.

OBJECTIVE: This study was undertaken to investigate the gut microbiome in progressive multiple sclerosis (MS) and how it relates to clinical disease. METHODS: We sequenced the microbiota from healthy controls and relapsing-remitting MS (RRMS) and progressive MS patients and correlated the levels of bacteria with clinical features of disease, including Expanded Disability Status Scale (EDSS), quality of life, and brain magnetic resonance imaging lesions/atrophy. We colonized mice with MS-derived Akkermansia and induced experimental autoimmune encephalomyelitis (EAE). RESULTS: Microbiota ß-diversity differed between MS patients and controls but did not differ between RRMS and progressive MS or differ based on disease-modifying therapies. Disease status had the greatest effect on the microbiome ß-diversity, followed by body mass index, race, and sex. In both progressive MS and RRMS, we found increased Clostridium bolteae, Ruthenibacterium lactatiformans, and Akkermansia and decreased Blautia wexlerae, Dorea formicigenerans, and Erysipelotrichaceae CCMM. Unique to progressive MS, we found elevated Enterobacteriaceae and Clostridium g24 FCEY and decreased Blautia and Agathobaculum. Several Clostridium species were associated with higher EDSS and fatigue scores. Contrary to the view that elevated Akkermansia in MS has a detrimental role, we found that Akkermansia was linked to lower disability, suggesting a beneficial role. Consistent with this, we found that Akkermansia isolated from MS patients ameliorated EAE, which was linked to a reduction in RORγt+ and IL-17-producing γδ T cells. INTERPRETATION: Whereas some microbiota alterations are shared in relapsing and progressive MS, we identified unique bacteria associated with progressive MS and clinical measures of disease. Furthermore, elevated Akkermansia in MS may be a compensatory beneficial response in the MS microbiome. ANN NEUROL 2021;89:1195-1211.

Alterations of the gut ecological and functional microenvironment in different stages of multiple sclerosis.

Multiple sclerosis (MS), an autoimmune disease of the central nervous system, generally starts as the relapsing remitting form (RRMS), but often shifts into secondary progressive MS (SPMS). SPMS represents a more advanced stage of MS, characterized by accumulating disabilities and refractoriness to medications. The aim of this study was to clarify the microbial and functional differences in gut microbiomes of the different stages of MS. Here, we compared gut microbiomes of patients with RRMS, SPMS, and two closely related disorders with healthy controls (HCs) by 16S rRNA gene and whole metagenomic sequencing data from fecal samples and by fecal metabolites. Each patient group had a number of species having significant changes in abundance in comparison with HCs, including short-chain fatty acid (SCFA)-producing bacteria reduced in MS. Changes in some species had close association with clinical severity of the patients. A marked reduction in butyrate and propionate biosynthesis and corresponding metabolic changes were confirmed in RRMS compared with HCs. Although bacterial composition analysis showed limited differences between the patient groups, metagenomic functional data disclosed an increase in microbial genes involved in DNA mismatch repair in SPMS as compared to RRMS. Together with an increased ratio of cysteine persulfide to cysteine in SPMS revealed by sulfur metabolomics, we postulate that excessive DNA oxidation could take place in the gut of SPMS. Thus, gut ecological and functional microenvironments were significantly altered in the different stages of MS. In particular, reduced SCFA biosynthesis in RRMS and elevated oxidative level in SPMS were characteristic.

Deep DNA metagenomic sequencing reveals oral microbiome divergence between monozygotic twins discordant for multiple sclerosis severity.

In contrast to gut, the oral microbiome of MS patients has not been characterized. Deep sequencing of saliva DNA from a pair of monozygotic twins (MSF1 with relapsing remitting MS; MSF2 with clinically isolated syndrome) identified 2036 bacterial species. Relative abundances of 3 phyla were higher, and 3 lower in MSF1 versus MSF2. Species diversity was greater in MSF2, and 20 abundant species differed at least 2-fold. Pathway analysis identified 116 functional hierarchies differing 50% or more. Although limited to one pair of twins, our data suggests that oral microbiome analysis may be useful for diagnosis or monitoring therapeutic efficacy.

Gut microbiome variation is associated to Multiple Sclerosis phenotypic subtypes.

OBJECTIVE: Multiple sclerosis (MS) is a heterogenous, inflammatory disease of the central nervous system. Microbiota alterations in MS versus healthy controls (HC) are observed, but results are inconsistent. We studied diversity, enterotypes, and specific gut microbial taxa variation between MS and HC, and between MS subgroups. METHODS: Amplicon sequencing of the 16S ribosomal RNA V4 region (Illumina MiSeq) was used to evaluate alpha and beta diversity, enterotypes, and relative taxa abundances on stool samples. MS subgroups were based on phenotype, disease course modifiers, and treatment status. Results were controlled for recently identified confounders of microbiota composition. RESULTS: Ninety-eight MS patients and 120 HC were included. Microbial richness was lower in interferon-treated (RRMS_I, N = 24) and untreated relapsing-remitting MS during relapse (RRMS_R, N = 4) when compared to benign (BMS, N = 20; Z = -3.07, Pcorr = 0.032 and Z = -2.68, Pcorr = 0.055) and primary progressive MS (PPMS, N = 26; Z = -2.39, Pcorr = 0.062 and Z = -2.26, Pcorr = 0.071). HC (N = 120) and active untreated MS (RRMS_U, N = 24) showed intermediate microbial richness. Enterotypes were associated with clinical subgroups (N = 218, χ2  = 36.10, P = 0.002), with Bacteroides 2 enterotype being more prevalent in RRMS_I. Butyricicoccus abundance was lower in PPMS than in RRMS_U (Z = -3.00, Pcorr = 0.014) and BMS (Z = -2.56, Pcorr = 0.031), lower in RRMS_I than in BMS (Z = -2.50, Pcorr = 0.034) and RRMS_U (Z = -2.91, Pcorr = 0.013), and inversely correlated with self-reported physical symptoms (rho = -0.400, Pcorr = 0.001) and disease severity (rho = -0.223, P = 0.027). INTERPRETATION: These results emphasize the importance of phenotypic subcategorization in MS-microbiome research, possibly explaining previous result heterogeneity, while showing the potential for specific microbiome-based biomarkers for disease activity and severity.

Disease-modifying therapies alter gut microbial composition in MS.

Objective: To determine the effects of the disease-modifying therapies, glatiramer acetate (GA) and dimethyl fumarate (DMF), on the gut microbiota in patients with MS. Methods: Participants with relapsing MS who were either treatment-naive or treated with GA or DMF were recruited. Peripheral blood mononuclear cells were immunophenotyped. Bacterial DNA was extracted from stool, and amplicons targeting the V4 region of the bacterial/archaeal 16S rRNA gene were sequenced (Illumina MiSeq). Raw reads were clustered into Operational Taxonomic Units using the GreenGenes database. Differential abundance analysis was performed using linear discriminant analysis effect size. Phylogenetic investigation of communities by reconstruction of unobserved states was used to investigate changes to functional pathways resulting from differential taxon abundance. Results: One hundred sixty-eight participants were included (treatment-naive n = 75, DMF n = 33, and GA n = 60). Disease-modifying therapies were associated with changes in the fecal microbiota composition. Both therapies were associated with decreased relative abundance of the Lachnospiraceae and Veillonellaceae families. In addition, DMF was associated with decreased relative abundance of the phyla Firmicutes and Fusobacteria and the order Clostridiales and an increase in the phylum Bacteroidetes. Despite the different changes in bacterial taxa, there was an overlap between functional pathways affected by both therapies. Interpretation: Administration of GA or DMF is associated with differences in gut microbial composition in patients with MS. Because those changes affect critical metabolic pathways, we hypothesize that our findings may highlight mechanisms of pathophysiology and potential therapeutic intervention requiring further investigation.

Infectious agents and different course of multiple sclerosis: a systematic review.

Multiple sclerosis (MS) causes demyelination of white matter of central nervous system and neuro-degeneration due to inflammation. Different types of MS, as well as disease progression, come with different pathology and pathophysiology. The objective of this study was to evaluate the possible association between different micro-organisms and the relapse or progression of MS. Studies indexed in Medline/PMC, Scopus and Web of Science published without time and language limitation until March 2017 were identified through the search terms "infection" or "infectious" and "multiple sclerosis". A total of 20878 abstracts were identified through the initial search terms. Selection of articles and assessment of their quality was done based on Cochrane library guidelines. Full texts were reviewed for 33 articles out of which 14 articles met the criteria for inclusion. Different micro-organisms are known to play roles in the pathogenesis of MS and its relapse; including Human herpesvirus 6 (HHV-6), Human herpesvirus 7 (HHV-7), Epstein-Barr virus (EBV), Chlamydia pneumoniae and Torque teno virus (TTV). But in this review only HHV-6, C. pneumoniae and TTV have been considered to play a role in disease progression in some studies and not all of them. This review concluded that some micro-organisms such as HHV-6, C. pneumoniae and TTV have been considered as cofactors to make MS a progressive type. It should be considered that these findings do not necessarily rule out the role of other pathogens in MS progression but may represent population differences or different sensitivity of the technique used.

Investigation of probiotics in multiple sclerosis.

None of the disease-modifying therapies (DMTs) currently being used for the management of multiple sclerosis (MS) are 100% effective. In addition, side effects associated with the use of these DMTs have limited the practice of combination therapy. Hence, there is a need for safe immunomodulatory agents to fine-tune the management of MS. The gut microbiome plays an important role in autoimmunity, and several studies have reported alterations in the gut microbiome of MS patients. Studies in animal model of MS have identified members of the gut commensal microflora that exacerbate or ameliorate neuroinflammation. Probiotics represent an oral, non-toxic immunomodulatory agent that could be used in combination with current MS therapy. We designed a pilot study to investigate the effect of VSL3 on the gut microbiome and peripheral immune system function in healthy controls and MS patients. VSL3 administration was associated with increased abundance of many taxa with enriched taxa predominated by Lactobacillus, Streptococcus, and Bifidobacterium species. At the immune level, VSL3 administration induced an anti-inflammatory peripheral immune response characterized by decreased frequency of intermediate monocytes (CD14highCD16low), decreased mean fluorescence intensity (MFI) of CD80 on classical monocytes as well as decreased human leukocyte antigen-antigen D related (HLA-DR) MFI on dendritic cells.

Are the decrease in circulating anti-α1,3-Gal IgG and the lower content of galactosyl transferase A1 in the microbiota of patients with multiple sclerosis a novel environmental risk factor for the disease?

The etiology of multiple sclerosis (MS), particularly the environmental component of the disease, remains speculative. Recent reports have suggested that alterations in the gut microbiota of MS patients could contribute to the etiology or pathophysiology of the disease. In this Viewpoint, using PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) to infer the functional content of the gut microbiota, we show that the gut microbiota of MS patients is characterized by a significant decrease in the relative abundance of the enzyme EC 2.4.1.87, which corresponds to the GGTA1 gene (which codes for the α1,3-Gal epitope and is lacking in humans), against which MS patients also have low levels of IgG antibodies. The decrease in circulating anti-α1,3-Gal IgG and lower content of galactosyl transferase A1 in the microbiota of patients with multiple sclerosis could be a novel environmental risk factor for the disease.

Intestinal Permeability in Relapsing-Remitting Multiple Sclerosis.

Changes of intestinal permeability (IP) have been extensively investigated in inflammatory bowel diseases (IBD) and celiac disease (CD), underpinned by a known unbalance between microbiota, IP and immune responses in the gut. Recently the influence of IP on brain function has greatly been appreciated. Previous works showed an increased IP that preceded experimental autoimmune encephalomyelitis development and worsened during disease with disruption of TJ. Moreover, studying co-morbidity between Crohn's disease and MS, a report described increased IP in a minority of cases with MS. In a recent work we found that an alteration of IP is a relatively frequent event in relapsing-remitting MS, with a possible genetic influence on the determinants of IP changes (as inferable from data on twins); IP changes included a deficit of the active mechanism of absorption from intestinal lumen. The results led us to hypothesize that gut may contribute to the development of MS, as suggested by another previous work of our group: a population of CD8+CD161high T cells, belonging to the mucosal-associated invariant T (MAIT) cells, a gut- and liver-homing subset, proved to be of relevance for MS pathogenesis. We eventually suggest future lines of research on IP in MS: studies on IP changes in patients under first-line oral drugs may result useful to improve their therapeutic index; correlating IP and microbiota changes, or IP and blood-brain barrier changes may help clarify disease pathogenesis; exploiting the IP data to disclose co-morbidities in MS, especially with CD and IBD, may be important for patient care.

Alterations of the human gut microbiome in multiple sclerosis.

The gut microbiome plays an important role in immune function and has been implicated in several autoimmune disorders. Here we use 16S rRNA sequencing to investigate the gut microbiome in subjects with multiple sclerosis (MS, n=60) and healthy controls (n=43). Microbiome alterations in MS include increases in Methanobrevibacter and Akkermansia and decreases in Butyricimonas, and correlate with variations in the expression of genes involved in dendritic cell maturation, interferon signalling and NF-kB signalling pathways in circulating T cells and monocytes. Patients on disease-modifying treatment show increased abundances of Prevotella and Sutterella, and decreased Sarcina, compared with untreated patients. MS patients of a second cohort show elevated breath methane compared with controls, consistent with our observation of increased gut Methanobrevibacter in MS in the first cohort. Further study is required to assess whether the observed alterations in the gut microbiome play a role in, or are a consequence of, MS pathogenesis.

Gut microbiota in early pediatric multiple sclerosis: a case-control study.

BACKGROUND AND PURPOSE: Alterations in the gut microbial community composition may be influential in neurological disease. Microbial community profiles were compared between early onset pediatric multiple sclerosis (MS) and control children similar for age and sex. METHODS: Children ≤18 years old within 2 years of MS onset or controls without autoimmune disorders attending a University of California, San Francisco, USA, pediatric clinic were examined for fecal bacterial community composition and predicted function by 16S ribosomal RNA sequencing and phylogenetic reconstruction of unobserved states (PICRUSt) analysis. Associations between subject characteristics and the microbiota, including beta diversity and taxa abundance, were identified using non-parametric tests, permutational multivariate analysis of variance and negative binomial regression. RESULTS: Eighteen relapsing-remitting MS cases and 17 controls (mean age 13 years; range 4-18) were studied. Cases had a short disease duration (mean 11 months; range 2-24) and half were immunomodulatory drug (IMD) naïve. Whilst overall gut bacterial beta diversity was not significantly related to MS status, IMD exposure was (Canberra, P < 0.02). However, relative to controls, MS cases had a significant enrichment in relative abundance for members of the Desulfovibrionaceae (Bilophila, Desulfovibrio and Christensenellaceae) and depletion in Lachnospiraceae and Ruminococcaceae (all P and q < 0.000005). Microbial genes predicted as enriched in MS versus controls included those involved in glutathione metabolism (Mann-Whitney, P = 0.017), findings that were consistent regardless of IMD exposure. CONCLUSIONS: In recent onset pediatric MS, perturbations in the gut microbiome composition were observed, in parallel with predicted enrichment of metabolic pathways associated with neurodegeneration. Findings were suggestive of a pro-inflammatory milieu.

Gut microbiota composition and relapse risk in pediatric MS: A pilot study.

We explored the association between baseline gut microbiota (16S rRNA biomarker sequencing of stool samples) in 17 relapsing-remitting pediatric MS cases and risk of relapse over a mean 19.8 months follow-up. From the Kaplan-Meier curve, 25% relapsed within an estimated 166 days from baseline. A shorter time to relapse was associated with Fusobacteria depletion (p=0.001 log-rank test), expansion of the Firmicutes (p=0.003), and presence of the Archaea Euryarchaeota (p=0.037). After covariate adjustments for age and immunomodulatory drug exposure, only absence (vs. presence) of Fusobacteria was associated with relapse risk (hazard ratio=3.2 (95% CI: 1.2-9.0), p=0.024). Further investigation is warranted. Findings could offer new targets to alter the MS disease course.

Mycoplasma pneumonia seroposivity in Iranian patients with relapsing-remitting multipl sclerosis: a randomized case-control study.

OBJECTIVES: Environmental factors, such as different infections, have proposed to be involved in the pathogenesis of multiple sclerosis (MS). This study aimed to the evaluate mycoplasma pneumonia seropositivity, as a common cause of community-acquired pneumonia in patients with relapsing-remitting multiple sclerosis (RRMS). METHODOLOGY: Using ELISA method, IgM and IgG antibodies to Mycoplasma pneumoniae were determined in 130 patients with relapsing-remitting multiple sclerosis (85 Remitted and 45 Relapsed) and 50 sex- and age-matched controls. The groups were compared using Kruskal-Wallis test at the significant level of p < 0.05. RESULTS: The median [interquartile range] titer of IgG in remitted multiple sclerosis group was 65.3 [51.1-75.2] RU/ml versus 64 [52.6-71.4] RU/ml in relapsed group and 57.5 [29.2-74.3] RU/ml in control group (p = 0.442). There was not any significant difference between the groups base on median titer of IgM too (p = 0.446). The median [interquartile range] titer of Mycoplasma pneumoniae (MPn) IgG in women was 69.2 [56.4-77.4] RU/ml in remitted patients versus 63.85 [52.45-71.25] RU/ml in relapsed patients and 55.2 [29.17-72.75] RU/ml in controls (p = 0.022). Post hoc analysis demonstrated significant difference between remitted patients and controls (p = 0.002). There was not any significant difference between men in the groups (p = 0.7). CONCLUSIONS: Mycoplasma seroposivity in relapsing-remitting multiple sclerosis was not significantly different in various phases of activity of disease compare to controls; but in women, seroposivity of Mycoplasma antibodies were more than controls.

Intracellular bacteria can cause EAE in SJL mice or modify self-specific T cell repertoire.

Environment and genetic are both relevant in determining development of Multiple Sclerosis. Many epidemiological observations converge on indicating EBV infection and Vitamin D levels as major players among the environmental factors. Bacteria and bacterial products are however potent triggers of immune responses, and recent work from several laboratories indicates that the microbiota plays a prominent role in "priming" or protecting individuals for development of experimental autoimmune diseases. Here we report our recent work dealing with the role of non-pathogenic mycobacteria and their innate receptors in relapsing-remitting experimental autoimmune encephalomyelitis in the SJL mouse and in mobilization of CNS-reactive T cells. We finally discuss how bacteria are likely involved in the pathogenesis of Multiple Sclerosis, expecially with regard to their role in driving the recurring acute episodes of disease.

Temporal relationship between environmental influenza A and Epstein-Barr viral infections and high multiple sclerosis relapse occurrence.

BACKGROUND: Multiple sclerosis (MS) relapses have been associated with viral and bacterial infection epidemics in MS patients who have not used interferon. OBJECTIVES: We studied whether environmental viral infections in the general population can be associated with increased MS relapse occurrence using retrospective data from 1986 to 1995 when interferons were not yet available. METHODS: Logistic regression modelling was used to compare retrospectively the monthly relapse occurrence from 407 MS patients in Turku University hospital archives and data on ten different specifically diagnosed viral infection epidemics in the general population of Southwestern Finland from 1986 to 1995. The outcome was the odds ratio (OR) of very high relapse occurrence versus low relapse occurrence, or moderate versus low relapse occurrence. RESULTS: After a peak in diagnosed influenza A cases in the general population, the MS relapse occurrence was 6.5 times more likely to be very high (95% CI 1.8-24.0) and 7.1 times more likely to be moderately high (95% CI 1.5-33.2). An increase in MS relapse counts also followed Epstein-Barr virus (EBV) infections (OR 4.4, 95% CI 1.3-15.1), but we found no significant association with adenovirus infections and MS relapses. The MS relapse occurrence was lowest in the summer months July-August (Chi-square test, p<0.01). CONCLUSIONS: Our findings suggest that influenza A and EBV viral infections in the general population are associated with a higher occurrence of exacerbations in MS patients, and thus environmental infection data should be included in epidemiological models on MS relapses.

Molecular detection of Parachlamydia-like organisms in cerebrospinal fluid of patients with multiple sclerosis.

The presence of Chlamydia-like organism DNA was investigated by polymerase chain reaction (PCR) in cerebrospinal fluid (CSF) samples from 27 patients previously found positive for Chlamydia pneumoniae DNA: 12 with multiple sclerosis (MS), grouped according to clinical and magnetic resonance imaging (MRI) evidence of disease activity, 8 with other inflammatory neurological disorders and 7 with non-inflammatory neurological disorders. PCR evidence of Chlamydia-like organisms in CSF was observed only in two relapsing-remitting MS patients with clinical and MRI disease activity. These findings suggest a possible association between C. pneumoniae and Chlamydia-like organism brain infections as a cofactor in MS development.

Chlamydophila pneumoniae DNA and mRNA transcript levels in peripheral blood mononuclear cells and cerebrospinal fluid of patients with multiple sclerosis.

Chlamydophila pneumoniae DNA and mRNA transcripts were investigated by PCR and RT-PCR in fresh CSF and PBMC specimens co-cultured in Hep-2 cell lines and collected from 14 patients with definite RR MS and 19 patients with other inflammatory (OIND) and non-inflammatory (NIND) neurological controls. A positivity for C. pneumoniae DNA and mRNA was detected in CSF and PBMCs of 9 RR MS patients (64.2%) with evidence of disease activity, whereas only 3 controls were positive for Chlamydial DNA. These preliminary findings suggest that C. pneumoniae may occur in a persistent and metabolically active state at both peripheral and intrathecal levels in MS, but not in OIND and NIND.

Cerebrospinal fluid molecular demonstration of Chlamydia pneumoniae DNA is associated to clinical and brain magnetic resonance imaging activity in a subset of patients with relapsing-remitting multiple sclerosis.

To further explore the link between Chlamydia pneumoniae and multiple sclerosis (MS), we examined cerebrospinal fluid (CSF) samples from 71 patients with MS and from 72 patients suffering from other inflammatory neurological disorders (OIND) or noninflammatory neurological disorders (NIND). All samples were analysed by a touchdown nested polymerase chain reaction (n-PCR) for C. pneumoniae with primer sets which amplify target sequence genes encoding the major outer membrane protein (MOMP), the 16S rRNA and the Hsp-70 protein. A molecular study was also performed to evaluate genetic diversity among isolates of C. pneumoniae and to compare chlamydial sequences. PCR was found positive in 36.6% of total MS, in 28.1% of OIND and in 37.5% of NIND patients, without any statistical differences among the various groups examined. CSF PCR evidence of C. pneumoniae was significantly more frequent in relapsing-remitting (RR) than in secondary progressive (SP) (P < 0.001) and in primary progressive (PP)MS (P < 0.05), in clinically active than in clinically stable MS (P < 0.05) and in MRI active than in MRI inactive MS (P < 0.001). The analysis of CSF expression of each single C. pneumoniae-specific gene revealed that detectable levels of MOMP were significantly more frequent in MS patients with relapse (P < 0.05), whereas PCR positivity for MOMP and 16S rRNA genes were more represented in MS patients with clinical and MRI evidence of disease activity (P < 0.05). Similar rates for MOMP and 16S rRNA genes were detected in CSF of both MS patients and controls, whereas CSF PCR positivity for Hsp-70 gene was observed in only three active RR MS patients. Sequence analysis revealed significant homologies with C. pneumoniae compared to other Chlamydial spp. These findings confirm that the C. pneumoniae detection within the central nervous system (CNS) is not selectively restricted to MS, but accounts in a variety of neurological diseases. In addition, our results suggest that CSF C. pneumoniae-specific DNA detection can occur in a subset of MS patients with clinical and MRI active RR form in whom a C. pneumoniae brain chronic persistent infection may play a significant role in the development of disease.