Qualitative and quantitative detection of Chlamydophila pneumoniae DNA in cerebrospinal fluid from multiple sclerosis patients and controls.

A standardized molecular test for the detection of Chlamydophila pneumoniae DNA in cerebrospinal fluid (CSF) would assist the further assessment of the association of C. pneumoniae with multiple sclerosis (MS). We developed and validated a qualitative colorimetric microtiter plate-based PCR assay (PCR-EIA) and a real-time quantitative PCR assay (TaqMan) for detection of C. pneumoniae DNA in CSF specimens from MS patients and controls. Compared to a touchdown nested-PCR assay, the sensitivity, specificity, and concordance of the PCR-EIA assay were 88.5%, 93.2%, and 90.5%, respectively, on a total of 137 CSF specimens. PCR-EIA presented a significantly higher sensitivity in MS patients (p = 0.008) and a higher specificity in other neurological diseases (p = 0.018). Test reproducibility of the PCR-EIA assay was statistically related to the volumes of extract DNA included in the test (p = 0.033); a high volume, which was equivalent to 100 microl of CSF per reaction, yielded a concordance of 96.8% between two medical technologists running the test at different times. The TaqMan quantitative PCR assay detected 26 of 63 (41.3%) of positive CSF specimens that tested positive by both PCR-EIA and nested-PCR qualitative assays. None of the CSF specimens that were negative by the two qualitative PCR methods were detected by the TaqMan quantitative PCR. The PCR-EIA assay detected a minimum of 25 copies/ml C. pneumoniae DNA in plasmid-spiked CSF, which was at least 10 times more sensitive than TaqMan. These data indicated that the PCR-EIA assay possessed a sensitivity that was equal to the nested-PCR procedures for the detection of C. pneumoniae DNA in CSF. The TaqMan system may not be sensitive enough for diagnostic purposes due to the low C. pneumoniae copies existing in the majority of CSF specimens from MS patients.

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.

The investigation of Chlamydophila pneumoniae in patients with multiple sclerosis.

Totally 32 cerebrospinal fluid samples from Multiple sclerosis (MS) patients were collected. DNA was extracted by High Pure PCR Template Preparation Kit. Two genomic segments, outer membrane protein genes ompA and omp9, were targeted for the detection of C. pneumoniae DNA in the samples by PCR tests. To detect ompA, a nested-PCR assay was designed, whereas for omp9, a PCR-Enyzme immunoassay (PCR-EIA) depending on streptavidin-biotin capture and dig detection of the PCR products was performed. C. pneumoniae DNA was not detected by each assays in patient samples.

Recurrent optic neuritis associated with Chlamydia pneumoniae infection of the central nervous system.

It has been suggested that Chlamydia pneumoniae (C. pneumoniae) is involved in the pathogenesis of diverse diseases of the central nervous system (CNS), including multiple sclerosis. We report the case of a 12-year-old male with isolated recurrent optic neuritis and an associated CNS infection with C. pneumoniae. The patient presented with three attacks of optic neuritis within 5 months. A positive polymerase chain reaction for C. pneumoniae in the cerebrospinal fluid led to the diagnosis of a CNS infection with C. pneumoniae. After treatment with the antibiotic rifampicin, he experienced no further attacks during the follow-up period of 6 years. These findings suggest the possibility of a C. pneumoniae infection as a contributing factor or even causative event for the development of optic neuritis.

An MRI study of Chlamydia pneumoniae infection in Italian multiple sclerosis patients.

We amplified sequences of the Chlamydia pneumoniae (CP) major-outer membrane protein in the cerebrospinal fluid (CSF) from 23 of 107 (21.5%) relapsing-remitting or secondary progressive multiple sclerosis (MS) patients and two of 77 (2.6%) patients with other neurological diseases (OND) (P = 0.00022). CP+ patients showed magnetic resonance imaging (MRI) evidence of more active disease (P = 0.02) compared to CP- MS patients and tended to have an anticipation of age at disease onset (32.3 +/- 12 versus 28.5 +/- 10 years; P = ns) causing a longer disease duration (7.5 +/- 5 versus 4.4 +/- 4 years; P = 0.016) at the time of clinical evaluation. These findings, although indirectly, suggest that CP infection of the central nervous system (CNS) might affect disease course in a subgroup of MS patients.

Is Chlamydia pneumoniae found in spinal fluid samples from multiple sclerosis patients? Conflicting results.

Cerebrospinal fluid samples from controls and patients with multiple sclerosis (MS) were split and sent to laboratories with different experiences for the detection of Chlamydia pneumoniae by polymerase chain reaction. Vanderbilt investigators identified C. pneumoniae in the majority of patients with MS and uncommonly in controls. Laboratories at Johns Hopkins University, University of Umeå, and the Centers for Disease Control and Prevention did not identify C. pneumoniae in any of the samples. Conflicting reports of C. pneumoniae detection in the some samples from patents with MS highlight the need to exchange detection techniques among laboratories involved in this controversy.

Intrathecal antibody production against Chlamydia pneumoniae in multiple sclerosis is part of a polyspecific immune response.

Chronic intrathecal immunoglobulin (Ig) production is a hallmark of multiple sclerosis characterized by the presence of oligoclonal IgGs and, in addition, polyspecific recognition of different pathogens such as measles, rubella and herpes zoster virus. While the antigen specificity of the oligoclonal IgGs in multiple sclerosis is largely unknown, the oligoclonal IgGs arising during CNS infectious diseases are reactive against the specific pathogen. Recently, a link between Chlamydia pneumoniae and multiple sclerosis has been claimed. To test the possible role of C. pneumoniae in multiple sclerosis, we analysed (i) whether there is intrathecal IgG production against C. pneumoniae in multiple sclerosis and (ii) if the oligoclonal IgGs in the CSF of multiple sclerosis patients recognize C. pneumoniae. By studying paired serum-CSF samples from 120 subjects (definite multiple sclerosis, 46; probable multiple sclerosis, 12; other inflammatory neurological diseases, 35; other neurological diseases, 27) by enzyme-linked immunosorbent assay, we found that 24% of all patients with definite multiple sclerosis, but only 5% of patients with other inflammatory or non-inflammatory diseases, produced IgGs specific for C. pneumoniae intrathecally (definite multiple sclerosis versus other inflammatory neurological diseases: P = 0.027). The presence of intrathecal IgGs to C. pneumoniae was independent of the duration of disease and relatively stable over time. The major CSF oligoclonal IgG bands from multiple sclerosis patients with an intrathecal Ig production to C. pneumoniae did not react towards purified elementary bodies and reticulate bodies of C. pneumoniae on affinity-mediated immunoblot following isoelectric focusing (IEF-western blots). In contrast, the IgGs in the CSF of control patients with neuroborreliosis strongly reacted with their specific pathogen, Borrelia burgdorferi, by IEF-western blot analysis. Concomitant analysis of the CSF of 23 patients with a nested polymerase chain reaction for C. pneumoniae was negative in all cases. Together, our findings strongly suggest that the immune response to C. pneumoniae is part of a polyspecific intrathecal Ig production, as is commonly observed with other pathogens. This argues against a specific role for C. pneumoniae in multiple sclerosis.

PCR-based method for isolation and detection of Chlamydia pneumoniae DNA in cerebrospinal fluids.

Since current studies indicate the possible involvement of Chlamydia pneumoniae in the pathogenesis of multiple sclerosis (MS), demonstration of C. pneumoniae in the cerebrospinal fluid (CSF) of patients with MS is highly desirable. However, there is controversy concerning the detection of C. pneumoniae in CSFs from MS patients due to the lack of a standard protocol for extraction and detection of C. pneumoniae DNA. In this regard, we attempted to establish a highly effective extraction protocol for C. pneumoniae DNA from CSFs utilizing a commercial kit and a PCR detection method. The extraction and PCR detection protocol established in this study succeeded in detecting as few as 20 C. pneumoniae organisms in 200 microl of mock CSF. The use of this protocol to detect C. pneumoniae DNA in CSFs revealed that 68% of CSF samples obtained from patients with MS were positive (11 out of 16 samples) for chlamydia DNA. Thus, the protocol established here is sensitive enough to detect chlamydia DNA from CSFs and can be used by other laboratories for evaluation of the presence of chlamydiae in CSFs because the protocol is based on the use of a commercial kit.