Chlamydophila pneumoniae infection of the central nervous system in patients with multiple sclerosis
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《神经病学神经外科学杂志》
1 Microbiology Department, University College London Hospitals, London WC1E 6DB, UK
2 Microbiology, Hospital for Sick Children, Great Ormond St, London WC1N 3JH
3 Department of Infection Control National Hospital for Neurology and Neurosurgery, Queen Square, London WC1N 3BG
4 Institute of Neurology, Queen Square, London WC1N 3BG
Correspondence to:
Dr S J Furrows
Microbiology Department, University College London Hospitals, Cecil Fleming House, London WC1E 6DB, UK; SFurrows@aol.com
ABSTRACT
Background: Chlamydophila pneumoniae has been postulated as an aetiological agent in the pathophysiology of multiple sclerosis. Previous studies show conflicting results.
Objective: To investigate patients with multiple sclerosis and other neurological diseases for evidence of past or present infection with C pneumoniae.
Methods: 19 patients with multiple sclerosis and 29 with other neurological diseases were studied. Evidence was sought for past or present infection with C pneumoniae using polymerase chain reaction (PCR) and cell culture of cerebrospinal fluid (CSF), and enzyme linked immunosorbent assay and microimmunofluorescence of serum.
Results: C pneumoniae was grown from the CSF of one patient with multiple sclerosis. PCR was negative in all cases. Anti-chlamydial antibodies were detected in the same proportion in each group.
Conclusions: This study does not support the theory of an association between C pneumoniae and multiple sclerosis.
Keywords: multiple sclerosis; chlamydophila pneumoniae
Abbreviations: MIF, microimmunofluorescence; ELISA, enzyme-linked immunosorbent assay; ELONA, enzyme-linked oligonucleotide assay; PCR, polymerase chain reaction; RFLP, restriction fragment length polymorphism
Chlamydophila pneumoniae is an obligate intracellular pathogen primarily associated with respiratory disease. It is capable of causing persistent or latent infections. A possible association between C pneumoniae and multiple sclerosis has been investigated,1–21 with conflicting results. We investigated a group of 19 patients with multiple sclerosis and 29 control patients with other neurological diseases. We looked for evidence of past or present infection with C pneumoniae, using polymerase chain reaction (PCR) and cell culture in cerebrospinal fluid (CSF), and enzyme linked immunosorbent assay (ELISA) and microimmunofluorescence (MIF) in serum.
METHODS
The study group consisted of 48 consecutive patients undergoing routine lumbar punctures for diagnostic purposes. Nineteen had a clinical diagnosis of multiple sclerosis (mean age 50.7 years, 10/19 (52.6%) male); 29 had other neurological diseases (mean age 39.8 years, 17/29 (58.6%) male). Half the control group had inflammatory diseases such as encephalitis or neurosarcoidosis, and half had non-inflammatory diseases such as dementia or epilepsy. Full ethical approval was obtained before the study.
CSF was collected directly into processing tubes and cultured on the day of collection onto HL22 and HEp223 cell lines, using a modification of Sriram’s method.1 Cell monolayers were examined on day 7 and day 14 for chlamydial inclusions using the IMAGEN direct immunofluorescent kit (Dako). The positive isolate was identified by analysis of the omp2 gene.24
For PCR, DNA was extracted using a modification of the method of Sriram,1 with the addition of an internal control (50 elementary bodies of C trachomatis added per sample). Analysis was done using a sensitive and specific PCR-ELONA (enzyme linked oligonucleotide assay).24
Serum from all patients was assayed by ELISA for IgM and IgG antibodies to the family Chlamydiaceae (MEDAC Diagnostika; interpretation according to manufacturer’s instructions). Samples positive by ELISA were further tested using an in-house microimmunofluorescence (MIF) assay, a sensitive and specific assay for anti-chlamydial antibodies that can distinguish between serotypes. We assigned high cut off values in order to exclude non-specific results (cut off titre, 256 for IgG; 64 for IgM and IgA).
A review of published reports was carried out following a Medline search, using the search terms "C pneumoniae" and "multiple sclerosis."
RESULTS
Culture
One CSF sample of the 48 tested was culture positive for C pneumoniae. The CSF came from a patient with multiple sclerosis (see case report below). Direct immunofluorescent staining showed inclusion bodies in HL and HEp2 monolayers at seven days. The number of inclusions seen at the first passage was very scanty, but improved after several further passages. The positive culture result was confirmed by repeating the culture using an untouched aliquot of CSF from the same patient, which had been taken at the same time as the original sample and frozen at -70°C. C pneumoniae was again grown from the CSF. PCR-RFLP and DNA sequencing of the partial omp2 gene product confirmed the isolate as C pneumoniae.
PCR
C pneumoniae DNA was not detected by PCR-ELONA in any patient sample. All external negative and positive extraction PCR and ELONA controls were valid. The internal control was not detected in four samples, including the culture positive sample.
Serology
Nine of the 19 patients with multiple sclerosis (47.4%) were antibody positive by ELISA. Four of these nine (44%) were positive by MIF for antibodies to C pneumoniae. Fifteen of 29 control patients (51.7%) were antibody positive by ELISA. Six of these 15 (40%) were positive by MIF for antibodies to C pneumoniae. Antibodies to C pneumoniae were therefore detected in the same proportion in each group: four of 19 patients with multiple sclerosis (21%) and six of 29 control patients (21%).
Review of published reports
Results of the literature review are given in table 1.
Table 1 Review of published reports: summary of results of cerebrospinal fluid (CSF) polymerase chain reaction (PCR), culture, and serology
CASE REPORT
The patient whose CSF was culture positive for C pneumoniae was in the multiple sclerosis group of the study. He presented in October 1998, aged 20, with gradual onset of progressive ataxia. In March 1999 he developed weakness of the left arm and numbness of the face and trunk. In August 1999 he developed loss of sensation in the right leg. Magnetic resonance imaging of the head was consistent with multiple sclerosis. Subsequently, isoelectric focusing with immunofixation of the CSF showed the presence of intrathecally synthesised oligoclonal IgG bands. A diagnosis of clinically definitive relapsing-remitting multiple sclerosis was made, supported by radiological findings.
The patient was recruited into this study in December 1999. C pneumoniae was grown from his CSF. Anti-chlamydial IgG antibodies were detected by ELISA at a titre of 200. MIF confirmed the presence of IgG antibody to C pneumoniae. IgM was not detected by ELISA or MIF, indicating that the infection was not acute. IgA was not detected by MIF. The serological findings suggest a previous (not acute) infection with C pneumoniae.
DISCUSSION
Multiple sclerosis is considered to be the end point of an autoimmune process triggered by an environmental factor in susceptible individuals. Numerous agents have been suggested as the environmental trigger. Sriram’s original study in 1999,1 which appeared to show a convincing link between C pneumoniae infection and multiple sclerosis, met with great interest. Subsequent studies by other institutions failed to produce convincing evidence of C pneumoniae infection in patients with multiple sclerosis. In order to investigate the association shown by Sriram et al, we chose to use similar methodology, concentrating on CSF culture, CSF PCR, and serology.
The most striking result in this study is the culture of C pneumoniae from the CSF of a patient with multiple sclerosis. This is the first time it has been cultured from CSF in the United Kingdom, and is only the third clinical isolate from any site in the United Kingdom. Serology confirmed that the culture positive patient had had previous C pneumoniae infection. PCR was negative from this sample and all others. The PCR incorporated an internal control, which proved that 90% of individual assays were able to detect at least 125 genomes/ml CSF. As the routine extraction, external PCR, and ELONA controls were valid, a negative result has been given on all samples. However, samples with negative internal controls could be reported as "no result" as the level of sensitivity is unknown. The internal control was not detected in the culture positive sample, explaining the discrepancy between the positive culture and negative PCR result.
Our literature review showed that results of different studies were highly inconsistent. Following the original study by Sriram et al,1 most but not all studies failed to detect C pneumoniae in patients with multiple sclerosis. A study by Kaufman et al,20 in which the same samples were tested at four different centres, showed great variation in results between centres. Therefore these differences cannot be attributed solely to differences in study populations but may also reflect differences in the collection and storage of samples, the DNA extraction method, or the culture/PCR techniques.
Conclusions
While we were able to culture C pneumoniae from the CSF in one patient of 19 with multiple sclerosis, serology did not show any difference in exposure between the subject groups, and PCR was negative in all cases. While the isolate is a unique culture result in the United Kingdom, our data do not support the hypothesis that C pneumoniae is implicated in the pathogenesis of multiple sclerosis. However, the recent nurses’ health study21 suggests there may be a positive association between C pneumoniae infection and multiple sclerosis, particularly progressive multiple sclerosis. There is a need for well designed clinical and epidemiological studies to produce more direct evidence for a role of C pneumoniae infection in multiple sclerosis.
ACKNOWLEDGEMENTS
This project was supported by a generous grant from the MS Society of Great Britain and Northern Ireland. Our thanks to Hussan Salman for technical advice.
REFERENCES
Sriram S, Stratton CW, Yao S, et al. Chlamydia pneumoniae infection of the central nervous system in multiple sclerosis. Ann Neurol 1999;46:6–14.
Sriram S, Mitchell W, Stratton C. Multiple sclerosis associated with Chlamydia pneumoniae infection of the CNS. Neurology 1998;50:571–2.
Boman J, Roblin PM, Sundstrom P, et al. Failure to detect Chlamydia pneumoniae in the central nervous system of patients with MS. Neurology 2000;54:265.
Dong-Si T, Bendl C, Blaas S, et al. Chlamydia pneumoniae is frequently found in cerebrospinal fluid of patients with multiple sclerosis. Ann Neurol 2000;48:448.
Layh-Schmitt G, Bendl C, Hildt U, et al. Evidence for infection with Chlamydia pneumoniae in a subgroup of patients with multiple sclerosis. Ann Neurol 2000;47:652–5.
Li WP, Ming X, Cook S, et al. Chlamydia pneumoniae sequence frequently present in both MS and control spinal fluid . Neurol 2000;54 (suppl 3) :A165.
Morre S, De Groot CJA, Killestein J, et al. Is Chlamydia pneumoniae present in the central nervous system of multiple sclerosis patients? Ann Neurol 2000;48:397.
Parratt J, Tavendale R, Parratt D, et al. Does Chlamydia pneumoniae infection occur in multiple sclerosis? a preliminary investigation of cerebrospinal fluid. J Neurol Neurosurg Psychiatry 2000;69:422.
Poland SD, Rice GPA. Chlamydia pneumoniae and multiple sclerosis . Neurology 2000;54 (suppl 3) :A165.
Pucci E, Taus C, Cartechini E, et al. Lack of Chlamydia infection of the central nervous system in multiple sclerosis . Ann Neurol 2000;48:399.
Treib J, Haa? A, Stille W, et al. Multiple sclerosis and Chlamydia pneumoniae . Ann Neurol 2000;47:408.
Derfuss T, Gurkov R, Then Bergh F, et al. Intrathecal antibody production against Chlamydia pneumoniae in multiple sclerosis is part of a polyspecific immune response. Brain 2001;124:1325–35.
Gieffers J, Pohl D, Treib J, et al. Presence of Chlamydia pneumoniae DNA in the cerebral spinal fluid is a common phenomenon in a variety of neurological diseases and not restricted to multiple sclerosis. Ann Neurol 2001;49:585–9.
Krametter D, Niederwieser G, Berghold A, et al. Chlamydia pneumoniae in multiple sclerosis: humoral immune responses in serum and cerebrospinal fluid and correlation with disease activity marker. Mult Scler 2001;7:13–18.
Numazaki K, Chibar S. Failure to detect Chlamydia pneumoniae in the central nervous system of patients with multiple sclerosis. Neurology 2001;57:746.
Pincherle A, Blasi F, Filippi M, et al. Association between Chlamydia pneumoniae infection and clinical activity in multiple sclerosis . Neurology 2001;56 (suppl 3) :A450.
Saiz A, Marcos MA, Graus F, et al. No evidence of CNS infection with Chlamydia pneumoniae in patients with multiple sclerosis. J Neurol 2001;248:617–18.
Sotgiu S, Piana A, Pugliatti M, et al. Chlamydia pneumoniae in the cerebrospinal fluid of patients with multiple sclerosis and neurological controls. Mult Scler 2001;7:371–4.
Hao Q, Miyashita N, Matsui M, et al. Chlamydia pneumoniae infection associated with enhanced MRI spinal lesions in multiple sclerosis. Mult Scler 2002;8:436–40.
Kaufman M, Gaydos CA, Sriram S, et al. Is Chlamydia pneumoniae found in spinal fluid samples from multiple sclerosis patients? Conflicting results. Mult Scler 2002;8:289–94.
Munger KL, Peeling RW, Hernan MA, et al. Infection with Chlamydia pneumoniae and risk of multiple sclerosis. Epidemiology 2003;14:141–7.
Kuo C-C, Grayston JT. A sensitive cell line, HL cells, for isolation and propagation of Chlamydia pneumoniae strain TWAR. J Infect Dis 1990;162:755–8.
Roblin PM, Dumornay W, Hammerschlag MR. Use of HEp-2 cells for improved isolation and passage of Chlamydia pneumoniae. J Clin Microbiol 1992;30:1968–71.
Hartley JC, Kaye S, Stevenson S, et al. PCR detection and molecular identification of Chlamydiaceae species. J Clin Microbiol 2001;39:3072–9.(S J Furrows1, J C Hartley)
2 Microbiology, Hospital for Sick Children, Great Ormond St, London WC1N 3JH
3 Department of Infection Control National Hospital for Neurology and Neurosurgery, Queen Square, London WC1N 3BG
4 Institute of Neurology, Queen Square, London WC1N 3BG
Correspondence to:
Dr S J Furrows
Microbiology Department, University College London Hospitals, Cecil Fleming House, London WC1E 6DB, UK; SFurrows@aol.com
ABSTRACT
Background: Chlamydophila pneumoniae has been postulated as an aetiological agent in the pathophysiology of multiple sclerosis. Previous studies show conflicting results.
Objective: To investigate patients with multiple sclerosis and other neurological diseases for evidence of past or present infection with C pneumoniae.
Methods: 19 patients with multiple sclerosis and 29 with other neurological diseases were studied. Evidence was sought for past or present infection with C pneumoniae using polymerase chain reaction (PCR) and cell culture of cerebrospinal fluid (CSF), and enzyme linked immunosorbent assay and microimmunofluorescence of serum.
Results: C pneumoniae was grown from the CSF of one patient with multiple sclerosis. PCR was negative in all cases. Anti-chlamydial antibodies were detected in the same proportion in each group.
Conclusions: This study does not support the theory of an association between C pneumoniae and multiple sclerosis.
Keywords: multiple sclerosis; chlamydophila pneumoniae
Abbreviations: MIF, microimmunofluorescence; ELISA, enzyme-linked immunosorbent assay; ELONA, enzyme-linked oligonucleotide assay; PCR, polymerase chain reaction; RFLP, restriction fragment length polymorphism
Chlamydophila pneumoniae is an obligate intracellular pathogen primarily associated with respiratory disease. It is capable of causing persistent or latent infections. A possible association between C pneumoniae and multiple sclerosis has been investigated,1–21 with conflicting results. We investigated a group of 19 patients with multiple sclerosis and 29 control patients with other neurological diseases. We looked for evidence of past or present infection with C pneumoniae, using polymerase chain reaction (PCR) and cell culture in cerebrospinal fluid (CSF), and enzyme linked immunosorbent assay (ELISA) and microimmunofluorescence (MIF) in serum.
METHODS
The study group consisted of 48 consecutive patients undergoing routine lumbar punctures for diagnostic purposes. Nineteen had a clinical diagnosis of multiple sclerosis (mean age 50.7 years, 10/19 (52.6%) male); 29 had other neurological diseases (mean age 39.8 years, 17/29 (58.6%) male). Half the control group had inflammatory diseases such as encephalitis or neurosarcoidosis, and half had non-inflammatory diseases such as dementia or epilepsy. Full ethical approval was obtained before the study.
CSF was collected directly into processing tubes and cultured on the day of collection onto HL22 and HEp223 cell lines, using a modification of Sriram’s method.1 Cell monolayers were examined on day 7 and day 14 for chlamydial inclusions using the IMAGEN direct immunofluorescent kit (Dako). The positive isolate was identified by analysis of the omp2 gene.24
For PCR, DNA was extracted using a modification of the method of Sriram,1 with the addition of an internal control (50 elementary bodies of C trachomatis added per sample). Analysis was done using a sensitive and specific PCR-ELONA (enzyme linked oligonucleotide assay).24
Serum from all patients was assayed by ELISA for IgM and IgG antibodies to the family Chlamydiaceae (MEDAC Diagnostika; interpretation according to manufacturer’s instructions). Samples positive by ELISA were further tested using an in-house microimmunofluorescence (MIF) assay, a sensitive and specific assay for anti-chlamydial antibodies that can distinguish between serotypes. We assigned high cut off values in order to exclude non-specific results (cut off titre, 256 for IgG; 64 for IgM and IgA).
A review of published reports was carried out following a Medline search, using the search terms "C pneumoniae" and "multiple sclerosis."
RESULTS
Culture
One CSF sample of the 48 tested was culture positive for C pneumoniae. The CSF came from a patient with multiple sclerosis (see case report below). Direct immunofluorescent staining showed inclusion bodies in HL and HEp2 monolayers at seven days. The number of inclusions seen at the first passage was very scanty, but improved after several further passages. The positive culture result was confirmed by repeating the culture using an untouched aliquot of CSF from the same patient, which had been taken at the same time as the original sample and frozen at -70°C. C pneumoniae was again grown from the CSF. PCR-RFLP and DNA sequencing of the partial omp2 gene product confirmed the isolate as C pneumoniae.
PCR
C pneumoniae DNA was not detected by PCR-ELONA in any patient sample. All external negative and positive extraction PCR and ELONA controls were valid. The internal control was not detected in four samples, including the culture positive sample.
Serology
Nine of the 19 patients with multiple sclerosis (47.4%) were antibody positive by ELISA. Four of these nine (44%) were positive by MIF for antibodies to C pneumoniae. Fifteen of 29 control patients (51.7%) were antibody positive by ELISA. Six of these 15 (40%) were positive by MIF for antibodies to C pneumoniae. Antibodies to C pneumoniae were therefore detected in the same proportion in each group: four of 19 patients with multiple sclerosis (21%) and six of 29 control patients (21%).
Review of published reports
Results of the literature review are given in table 1.
Table 1 Review of published reports: summary of results of cerebrospinal fluid (CSF) polymerase chain reaction (PCR), culture, and serology
CASE REPORT
The patient whose CSF was culture positive for C pneumoniae was in the multiple sclerosis group of the study. He presented in October 1998, aged 20, with gradual onset of progressive ataxia. In March 1999 he developed weakness of the left arm and numbness of the face and trunk. In August 1999 he developed loss of sensation in the right leg. Magnetic resonance imaging of the head was consistent with multiple sclerosis. Subsequently, isoelectric focusing with immunofixation of the CSF showed the presence of intrathecally synthesised oligoclonal IgG bands. A diagnosis of clinically definitive relapsing-remitting multiple sclerosis was made, supported by radiological findings.
The patient was recruited into this study in December 1999. C pneumoniae was grown from his CSF. Anti-chlamydial IgG antibodies were detected by ELISA at a titre of 200. MIF confirmed the presence of IgG antibody to C pneumoniae. IgM was not detected by ELISA or MIF, indicating that the infection was not acute. IgA was not detected by MIF. The serological findings suggest a previous (not acute) infection with C pneumoniae.
DISCUSSION
Multiple sclerosis is considered to be the end point of an autoimmune process triggered by an environmental factor in susceptible individuals. Numerous agents have been suggested as the environmental trigger. Sriram’s original study in 1999,1 which appeared to show a convincing link between C pneumoniae infection and multiple sclerosis, met with great interest. Subsequent studies by other institutions failed to produce convincing evidence of C pneumoniae infection in patients with multiple sclerosis. In order to investigate the association shown by Sriram et al, we chose to use similar methodology, concentrating on CSF culture, CSF PCR, and serology.
The most striking result in this study is the culture of C pneumoniae from the CSF of a patient with multiple sclerosis. This is the first time it has been cultured from CSF in the United Kingdom, and is only the third clinical isolate from any site in the United Kingdom. Serology confirmed that the culture positive patient had had previous C pneumoniae infection. PCR was negative from this sample and all others. The PCR incorporated an internal control, which proved that 90% of individual assays were able to detect at least 125 genomes/ml CSF. As the routine extraction, external PCR, and ELONA controls were valid, a negative result has been given on all samples. However, samples with negative internal controls could be reported as "no result" as the level of sensitivity is unknown. The internal control was not detected in the culture positive sample, explaining the discrepancy between the positive culture and negative PCR result.
Our literature review showed that results of different studies were highly inconsistent. Following the original study by Sriram et al,1 most but not all studies failed to detect C pneumoniae in patients with multiple sclerosis. A study by Kaufman et al,20 in which the same samples were tested at four different centres, showed great variation in results between centres. Therefore these differences cannot be attributed solely to differences in study populations but may also reflect differences in the collection and storage of samples, the DNA extraction method, or the culture/PCR techniques.
Conclusions
While we were able to culture C pneumoniae from the CSF in one patient of 19 with multiple sclerosis, serology did not show any difference in exposure between the subject groups, and PCR was negative in all cases. While the isolate is a unique culture result in the United Kingdom, our data do not support the hypothesis that C pneumoniae is implicated in the pathogenesis of multiple sclerosis. However, the recent nurses’ health study21 suggests there may be a positive association between C pneumoniae infection and multiple sclerosis, particularly progressive multiple sclerosis. There is a need for well designed clinical and epidemiological studies to produce more direct evidence for a role of C pneumoniae infection in multiple sclerosis.
ACKNOWLEDGEMENTS
This project was supported by a generous grant from the MS Society of Great Britain and Northern Ireland. Our thanks to Hussan Salman for technical advice.
REFERENCES
Sriram S, Stratton CW, Yao S, et al. Chlamydia pneumoniae infection of the central nervous system in multiple sclerosis. Ann Neurol 1999;46:6–14.
Sriram S, Mitchell W, Stratton C. Multiple sclerosis associated with Chlamydia pneumoniae infection of the CNS. Neurology 1998;50:571–2.
Boman J, Roblin PM, Sundstrom P, et al. Failure to detect Chlamydia pneumoniae in the central nervous system of patients with MS. Neurology 2000;54:265.
Dong-Si T, Bendl C, Blaas S, et al. Chlamydia pneumoniae is frequently found in cerebrospinal fluid of patients with multiple sclerosis. Ann Neurol 2000;48:448.
Layh-Schmitt G, Bendl C, Hildt U, et al. Evidence for infection with Chlamydia pneumoniae in a subgroup of patients with multiple sclerosis. Ann Neurol 2000;47:652–5.
Li WP, Ming X, Cook S, et al. Chlamydia pneumoniae sequence frequently present in both MS and control spinal fluid . Neurol 2000;54 (suppl 3) :A165.
Morre S, De Groot CJA, Killestein J, et al. Is Chlamydia pneumoniae present in the central nervous system of multiple sclerosis patients? Ann Neurol 2000;48:397.
Parratt J, Tavendale R, Parratt D, et al. Does Chlamydia pneumoniae infection occur in multiple sclerosis? a preliminary investigation of cerebrospinal fluid. J Neurol Neurosurg Psychiatry 2000;69:422.
Poland SD, Rice GPA. Chlamydia pneumoniae and multiple sclerosis . Neurology 2000;54 (suppl 3) :A165.
Pucci E, Taus C, Cartechini E, et al. Lack of Chlamydia infection of the central nervous system in multiple sclerosis . Ann Neurol 2000;48:399.
Treib J, Haa? A, Stille W, et al. Multiple sclerosis and Chlamydia pneumoniae . Ann Neurol 2000;47:408.
Derfuss T, Gurkov R, Then Bergh F, et al. Intrathecal antibody production against Chlamydia pneumoniae in multiple sclerosis is part of a polyspecific immune response. Brain 2001;124:1325–35.
Gieffers J, Pohl D, Treib J, et al. Presence of Chlamydia pneumoniae DNA in the cerebral spinal fluid is a common phenomenon in a variety of neurological diseases and not restricted to multiple sclerosis. Ann Neurol 2001;49:585–9.
Krametter D, Niederwieser G, Berghold A, et al. Chlamydia pneumoniae in multiple sclerosis: humoral immune responses in serum and cerebrospinal fluid and correlation with disease activity marker. Mult Scler 2001;7:13–18.
Numazaki K, Chibar S. Failure to detect Chlamydia pneumoniae in the central nervous system of patients with multiple sclerosis. Neurology 2001;57:746.
Pincherle A, Blasi F, Filippi M, et al. Association between Chlamydia pneumoniae infection and clinical activity in multiple sclerosis . Neurology 2001;56 (suppl 3) :A450.
Saiz A, Marcos MA, Graus F, et al. No evidence of CNS infection with Chlamydia pneumoniae in patients with multiple sclerosis. J Neurol 2001;248:617–18.
Sotgiu S, Piana A, Pugliatti M, et al. Chlamydia pneumoniae in the cerebrospinal fluid of patients with multiple sclerosis and neurological controls. Mult Scler 2001;7:371–4.
Hao Q, Miyashita N, Matsui M, et al. Chlamydia pneumoniae infection associated with enhanced MRI spinal lesions in multiple sclerosis. Mult Scler 2002;8:436–40.
Kaufman M, Gaydos CA, Sriram S, et al. Is Chlamydia pneumoniae found in spinal fluid samples from multiple sclerosis patients? Conflicting results. Mult Scler 2002;8:289–94.
Munger KL, Peeling RW, Hernan MA, et al. Infection with Chlamydia pneumoniae and risk of multiple sclerosis. Epidemiology 2003;14:141–7.
Kuo C-C, Grayston JT. A sensitive cell line, HL cells, for isolation and propagation of Chlamydia pneumoniae strain TWAR. J Infect Dis 1990;162:755–8.
Roblin PM, Dumornay W, Hammerschlag MR. Use of HEp-2 cells for improved isolation and passage of Chlamydia pneumoniae. J Clin Microbiol 1992;30:1968–71.
Hartley JC, Kaye S, Stevenson S, et al. PCR detection and molecular identification of Chlamydiaceae species. J Clin Microbiol 2001;39:3072–9.(S J Furrows1, J C Hartley)