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PCR for bacteria in multiple sclerosis cerebrospinal fluid
J.W. Lindsey, S. Patel (Houston, USA)

Objective: The objective of this study was to use a sensitive PCR method to test for the presence of seven different groups of bacteria in the cerebrospinal fluid (CSF) of multiple sclerosis patients.

Background: The etiology of multiple sclerosis is currently unknown, but many features of the disease suggest an infectious cause. The clinical course, the inflammatory infiltrates seen on histology, and the oligoclonal bands in the cerebrospinal fluid are all consistent with a recurrent or persistent infection. Similar diseases, such as acute transverse myelitis, acute disseminated encephalomyelitis, and Guillain-Barre syndrome, are linked to infections. And chronic infection with Treponema or Borrelia can cause neurologic disease.

Methods: We designed nested sets of PCR primers specific for the 16S ribosomal DNA of spirochetes, campylobacter, mycoplasma, chlamydia, bartonella, mycobacteria, and streptococcus. Each set of primers is designed to amplify DNA from all members of the desired group without amplifying DNA from common laboratory contaminants. We extracted DNA from the CSF of 10 patients with relapsing-remitting MS, 10 patients with primary progressive MS, and 9 controls. We amplified the DNA with nested PCR, and visualized the PCR products on agarose gels. Each experiment included both MS patients, controls, and a saline negative control. We defined the sensitivity of our method using serial dilutions of known amounts of bacterial DNA.

Results: The majority of the CSF specimens were negative for bacteria with all sets of primers. Three MS specimens and one control had a faint band of the correct size with the spirochete primers. Sequencing identified this PCR product as coming from a propionibacterium rather than a spirochete. Further experiments with primers specific for that sequence demonstrated that it was present at low concentrations in all specimens and is likely a laboratory contaminant. The sensitivity of the method was excellent. We could detect 10 copies or less of bacterial DNA per PCR reaction. This corresponds to a concentration of 200 bacteria per ml of CSF.
Conclusions: Using a very sensitive PCR method, we were unable to find evidence for the presence of any of the seven tested groups of bacteria in the CSF of MS patients.
This study supported in part by the National Multiple Sclerosis Society

Background: The etiology of multiple sclerosis is currently unknown, but many features of the disease suggest an infectious cause. The clinical course, the inflammatory infiltrates seen on histology, and the oligoclonal bands in the cerebrospinal fluid are all consistent with a recurrent or persistent infection. Similar diseases, such as acute transverse myelitis, acute disseminated encephalomyelitis, and Guillain-Barre syndrome, are linked to infections. And chronic infection with Treponema or Borrelia can cause neurologic disease

The most frequent and serious manifestation of disseminated Lyme borreliosis is neuroborreliosis. PCR was applied to 190 patients with untreated and confirmed neuroborreliosis. B. burgdorferi DNA was detectable in 17-21% of CSF samples from patients with neuroborreliosis. In patients with very early neuroborreliosis (< 2 weeks), still being negative for specific intrathecal antibody synthesis, a positive PCR was more frequent than in patients with longer disease duration.

no agent has yet emerged with any consensus as the cause of MS. This controversy is due to a number of factors, including lack of specificity of an agent to MS, lack of reproducibility in other laboratories, inappropriate controls, laboratory contamination and lack of a standard and easily reproducible assay system.

Chlamydia pneumoniae infection associated with enhanced MRI spinal lesions in multiple sclerosis.Hao Q, Miyashita N, Matsui M, Wang HY, Matsushima T, Saida T.
Department of Neurology, Center for Neurological Diseases, Utano National Hospital, Kyoto, Japan.

Cerebrospinal fluid (CSF) from 66 patients with multiple sclerosis (MS) and 25 patients with other neurological diseases (OND) were examined for the infection of Chlamydia pneumoniae by culture, polymerase chain reaction (PCR) assay, and determination of antibodies to C. pneumoniae. PCR was positive not only in 9 of 28 (32%) patients with MS but also in 2 patents with inflammatory disorders in 15 (13%) OND controls (p = 0.18). Viable C. pneumoniae was isolated from one patient with MS and one with paraneoplastic encephalomyelitis. C. pneumoniae could be detected only in cell-containing CSF. In MS, enhanced spinal magnetic resonance imaging (MRI) lesions were detected in all of four PCR-positive patents but none of five PCR-negative patients, and the difference was significant (p = 0.0079). However, no correlation was found between enhanced brain MRI lesions and CSF C. pneumoniae DNA. Elevated titers of anti-C. pneumoniae IgG were detected in CSF in 13 of 66 (20%) patients with MS and 1 of 25 (4%) OND controls (p = 0.064). CNS C. pneumoniae infection is not uncommon in MS as well as in other inflammatory disorders of the nervous system. The association of active spinal lesions with Chlamydia in CSF collected by lumber puncture suggests the detection of a recent infection. On the other hand, the lack of association of active MS brain lesions with CSF Chlamydia and the presence of PCR-positive patents who are clinically stable and have no enhancing MRI lesions imply the existence of a chronic infectious process.

PMID: 12356213 [PubMed - indexed for MEDLINE]

Hammerschlag MR.
Division of Pediatric Infectious Diseases, SUNY Downstate Medical Center, Brooklyn, NY 11203-2098, USA. mhammerschlag@pol.net

One of the major characteristics of Chlamydia spp. is its ability to cause prolonged, often subclinical infections. Chronic, persistent infection with Chlamydia pneumoniae has been implicated in the pathogenesis of several chronic diseases initially not thought to be infectious, including asthma, arthritis and atherosclerosis. C. pneumoniae is susceptible in vitro to a wide range of antimicrobial agents that target either protein or DNA synthesis, including macrolides, ketolides, tetracyclines, quinolones and rifamycins. Practically all treatment studies evaluating presented or published to date have used serology alone for diagnosis of C. pneumoniae infection, which only provides a clinical end point. The results of several treatment studies that did perform culture found that erythromycin, azithromycin (Zithromax, clarithromycin (Biaxin, levofloxacin (Levaquin and moxifloxacin (Avelox had a 70 to 90% efficacy in eradicating C. pneumoniae from the respiratory tract of children and adults with pneumonia. Persistence of the organism does not appear to be due to the development of antibiotic resistance. However, one cannot extrapolate from this experience to the treatment of chronic C. pneumoniae infection, especially cardiovascular disease. As there are no reliable serologic markers for chronic or persistent C. pneumoniae infection, it cannot be determined who is infected and who is not, which means that it cannot be assumed that any effect seen is due to successful treatment or eradication of C. pneumoniae.

PMID: 15482145 [PubMed - indexed for MEDLINE]