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.

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.

Efficacy of a novel reverse transcriptase-polymerase chain reaction (RT-PCR) for detecting Toxoplasma gondii bradyzoite gene expression in human clinical specimens.

A reverse transcriptase-polymerase chain reaction (RT-PCR) assay, was performed to evaluate the transcription degree of bradyzoite- or tachyzoite-specific genes of Toxoplasma gondii on cerebrospinal fluid (CSF) specimens from AIDS patients with toxoplasmic encephalitis (TE), and to distinguish an asymptomatic latent infection from a reactivated disease. This method was compared with nested DNA amplification (n)-PCR. The mRNA expression of the representative T. gondii cystic matrix (MAG1) or bradyzoite-specific (SAG4) genes was investigated on CSF obtained from AIDS patients with first episode (no. 11) or relapse (no. 8) of TE. The mRNA expression of tachyzoite-specific (SAG1) gene was also studied. New designed oligonucleotide primers and probes, which identify a 212 bp fragment inside to the open reading MAG1 sequence, were employed in both RT-PCR and n-PCR assays. Oligo-dT primed cDNA synthesis appeared a suitable method for subsequent analysis by n-PCR. RT-PCR has been shown to be more sensitive and specific than n-PCR. MAG1 and SAG4 gene expression was detected in 8 (100%) and 6 (75%) patients with TE relapses, respectively, while SAG1 detected 7 (63%) patients with TE first episode. These findings suggest that RT-PCR method is able to identify the bradyzoite stage of T. gondii especially in patients who are at risk for TE relapse.

Evidence of cerebrospinal fluid free kappa light chains in AIDS patients with Toxoplasma gondii encephalitis.

Cerebrospinal fluid (CSF) free light chains of kappa or lambda (FLC kappa/lambda) type were investigated by affinity mediated blotting technique (AMI) and ELISA in 28 patients of which nine with AIDS and Toxoplasma gondii encephalitis (AIDS, TE), 11 with AIDS with or without other CNS AIDS-related opportunistic infections (non-TE AIDS) and eight control patients with or without inflammatory neurological disorders (control group). CSF restricted oligoclonal FLC bands either of k or lambda isotype or both were found by AMI in 18 (90%) out of 20 AIDS patients, while a CSF pattern predominantly characterized by FkappaLC rather than FlambdaLC was observed in eight (88.8%) out of nine TE patients. No FLC components were detected in the matched sera of TE or non-TE AIDS patients or in the CSF and sera from control group. The anti-parasite-specific FkappaLC CSF/serum mean levels and the T. gondii-specific FkappaLC index values were found by ELISA to be significantly more elevated in TE patients when compared to non-TE AIDS or control group. These findings suggest that the increased production of T. gondii-specific FkappaLC could provide insights into pathogenesis of reactivated TE in immunocompromised patients and may have important diagnostic usefulness.