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Division of Infectious Diseases, Department of Pediatrics, State University of New York Downstate Medical Center, Brooklyn, Respiratory Diseases Branch, Division of Bacterial and Mycotic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia
Division of Infectious Diseases, Department of Medicine, Johns Hopkins University, Baltimore, Maryland
Department of Clinical Microbiology, Vienna General Hospital, Vienna, Austria
Department of Virology, Umea University, Umea, Sweden
The possible association between Chlamydia pneumoniae infection and multiple sclerosis (MS) was first described in a case study by Sriram and colleagues at Vanderbilt University Medical Center (VUMC), which was then followed by a study of a series of patients from VUMC in whom the researchers reported that the organism was identified by culture and polymerase chain reaction (PCR) [1, 2]. The results of subsequent studies performed by a number of other groups have been conflicting, finding C. pneumoniae DNA in 0% to >80% of cerebrospinal fluid (CSF) samples from patients with MS and in 0%20% of CSF samples from patients with other neurologic diseases [3]. This discrepancy in results is similar to inconsistencies in findings reported for the association between C. pneumoniae infection and atherosclerosis; some studies have identified the organism by PCR and/or immunohistochemical (IHC) staining in up to 100% of atheromatous tissues tested, but other studies have not confirmed these findings [4, 5]. These discrepancies may indicate methodologic differences, sampling error, or other unknown problems. Several multicenter studies have demonstrated major differences in methodologies and results, including significant inter- and intralaboratory variability in PCR testing when the same specimens were tested in different laboratories, even among those using the same assays [46].
The conflict between results reported in studies of the association between C. pneumoniae infection and MS may also be the result of methodologic problems. In 2002, Kaufman et al. [7], in an effort to deal with the issue of interlaboratory differences in methods used to detect C. pneumoniae in patients with MS, prospectively collected 30 CSF samples from patients with MS and 22 CSF samples from patients with other neurologic diseases; these samples were sent to laboratories at VUMC, Johns Hopkins University (JHU), and Umea University (UU) in Sweden and, subsequently, to the Centers for Disease Control and Prevention (CDC). None of the CSF samples was found to be positive by PCR at JHU, UU, and the CDC, but 73% of the CSF samples from patients with MS and 23% of the control samples were found to be positive by PCR at VUMC. Possible reasons for these discrepant results were discussed in the published article and included (1) poor sensitivities of the 3 different and well-validated PCR assays used by JHU, UU, and the CDC and (2) specificity problems with the PCR assay used by VUMC. Since the results of only 1 of 4 different PCRs from 1 laboratory were discrepant, of interest was the specificity of the primer sequences used in the VUMC PCRthat is, were they specific for only C. pneumoniae The primer sets used by VUMC in the multicenter study and the sets used in previous studies performed in that laboratory were analyzed and were found to have high sequence similarity to human DNA, as determined by BLAST search and amplification of human DNA [8]. These findings suggested that the primers used were not uniquely specific for C. pneumoniae.
The present article by Sriram et al. [9] should be read in this context. The authors report identification of C. pneumoniae in CSF and brain tissue from a high proportion of patients with MS, as detected by both nested PCR and IHC staining. Two different sets of PCR primers were used, directed at the C. pneumoniae major outer membrane protein (MOMP) and 16s RNA genes. However, the primer sets for MOMP used in the present study were the same as the ones VUMC used in the multicenter study and later analyzed [7, 8]. There are numerous issues regarding the specificity of the PCR used for the detection of C. pneumoniae in this study, as well as other PCR data generated by this method [8]. These issues include the specificity of the primer sequences, the low temperature used for the annealing reaction, and the questionable application of a nested touchdown assay format. Primers for both PCR assays used in the study target homologous human DNA sequences in addition to C. pneumoniae, according to BLAST as of January 2005, and, thus, may lead to nonspecific PCR product formation. Thus, it is likely that primers used in both PCRs generated nonspecific amplification products.
The format chosen for both PCR assays also poses a specificity problem. Both PCRs were based on the touchdown technique, which should use a high (e.g., 65°C) annealing temperature during the first cycles to increase specificity, followed by decreasing annealing temperatures for efficient amplicon amplification. However, the PCR used by Sriram et al. [9] began the touchdown protocol with annealing at 58°C, followed by decreases in temperature to 48°C. The above-described primer pairs in combination with this unexacting touchdown protocol may also lead to a nonspecific PCR product [8].
Considerable difficulty has also been experienced with antigen detection, because of nonspecific background staining and the morphological heterogeneity of C. pneumoniae elementary bodies [10]. Human and chlamydial heat-shock proteins have highly conserved and homologous antigenic sites. Consequently, such antigens may interfere in tissue diagnostics, resulting in cross-reactions and false-positive results [11]. In the Sriram et al. study, the authors report positive results based on IHC detection methods for 7 of 20 samples of formalin-fixed brain tissue from patients with MS, by use of 3 Chlamydia genus-specific antibodies, but the positive results could not be confirmed by staining with 2 different C. pneumoniae species-specific antibodies. The authors used mouse lungs infected with C. pneumoniae as a "positive" control. Although such infected mouse lung cells can be used to establish the sensitivity of the IHC assay, they cannot be used for establishing the specificity of an assay intended for detection of C. pneumoniae in human tissue. The detection of putative C. pneumoniae DNA and antigens more frequently in the patients with MS than in control subjects is also probably due to nonspecific DNA sequences that may be present more frequently in MS, including lymphocytes and other cells in the CSF and increased synthesis of immunoglobulins. The rate of detection of C. pneumoniae DNA in the control CSF samples is consistent with the background rate of contamination seen with nested PCR assays [6].
When in-houseproduced laboratory methods are developed and implemented, as in the study by Sriram et al., it is important that the investigators provide validation that the methods are specific and appropriate for the purpose for which they are being used. In summary, until more or new research studies indicate differently, we cannot scientifically support a biological association between C. pneumoniae infection and MS.
References
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