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Departments of Neurology and Pathology, Vanderbilt University Medical Center, Nashville, Tennessee
In their letter, Hammerschlag et al. [1] raise 2 major issues to which we would like to respond. These issues are the specificity of the polymerase chain reaction (PCR) assays and of the immunohistochemical (IHC) staining we used in our study [2].
The comments pertaining to the lack of specificity of the primer set and PCR amplification procedure for amplification of the major outer membrane protein (MOMP) gene are identical to those the same authors have raised previously; we have addressed them in the past [3]. There is no single accepted method for detection of Chlamydia pneumoniae DNA and, therefore, in-house assays that are verified internally are being used. We recognize the problems inherent in using nested PCR assays. To increase the specificity of our nucleic acidbased amplification assays, we required amplification with primers for 2 different C. pneumoniae genes to designate a sample as positive [4].
We must also point out that the conditions of our touchdown technique for the amplification of the 16s RNA gene began with an annealing temperature of 60°Cand not 58°C, as the authors indicateda temperature that was similar to the beginning temperature (62°C) in experiments published by the authors and not 65°C, as they now suggest [5].
Regarding the use of IHC staining, Hammerschlag et al. state that the initial positive control should be human tissue infected with C. pneumoniae. We have, however, followed the recommendation on IHC staining that 3 of the authors of the letter suggested in their review article and used infected tissue from experimental animals as our internal positive controls [6].
Hammerschlag et al. also comment that the antichlamydial hsp60 antibody that was used for IHC staining can cross-react with human hsp60 antigens, citing the work of Ochiai et al. to support their view [7]. The data in this article, however, do not support this assertion. The anti-hsp60 antibody that we used does not cross-react with human hsp60. The staining of chlamydial antigens obtained with the 3 different antibodies we used was similar in pattern and distribution and was not seen with the isotype-matched control antibodies. Thus, we believe our findings are unlikely to represent false-positive staining.
Hammerschlag and colleagues have, in prior studies, not detected C. pneumoniae in CNS tissue of patients with multiple sclerosis (MS), arguing for their absence in MS [8]. Their study did not anticipate the possibility that C. pneumoniae antigens are present predominantly in ependymal cells. Since their experimental design failed to address the possibility of distinct anatomic localization of Chlamydia antigens, their experiments proved unsuccessful. Furthermore, our findings showing an association between C. pneumoniae infection and MS are supported by the results of studies performed in laboratories in 4 different countries. At least 7 studies have shown C. pneumoniae DNA to be present in the CSF of patients with MS and absent in the CSF of control individuals [9]. The more recent finding of C. pneumoniae RNA in the CSF of patients with MS also supports our observation [10]. In addition, other laboratories have detected high-affinity antibodies to C. pneumoniae in the CSF [11]. Seroepidemiologic studies have shown an association between progression of MS and titers of antibody to C. pneumoniae [11, 12]. Thus, we argue that our data and those of others support an association between C. pneumoniae infection and MS.
References
1. Hammerschlag MR, Apfalter P, Boman J, Tondella ML, Gaydos C. The role of Chlamydia pneumoniae in multiple sclerosis: real or fictitious J Infect Dis 2005; 192:13057 (in this issue). First citation in article
2. Sriram S, Ljunggren-Rose A, Yao S-Y, Whetsell WO Jr. Detection of chlamydial bodies and antigens in the central nervous system of patients with multiple sclerosis. J Infect Dis 2005; 192:121928 (in this issue). First citation in article
3. Sriram S, Yao S, Stratton CS, Mitchell W, Yamamoto Y. Is C. pneumoniae present in CSF of MS patients Authors' reply. Clin Diagn Lab Immunol 2003; 10:978. First citation in article
4. Sriram S, Yao SY, Stratton C, et al. Comparative study of the presence of Chlamydia pneumoniae in cerebrospinal fluid of patients with clinically definite and monosymptomatic multiple sclerosis. Clin Diagn Lab Immunol 2002; 9:13327. First citation in article
5. Madico G, Quinn TC, Boman J, Gaydos CA. Touchdown enzyme time release-PCR for detection and identification of Chlamydia trachomatis, C. pneumoniae, and C. psittaci using the 16S and 16S-23S spacer rRNA genes. J Clin Microbiol 2000; 38:108593. First citation in article
6. Dowell SF, Peeling RW, Boman J, et al. Standardizing Chlamydia pneumoniae assays: recommendations from the Centers for Disease Control and Prevention (USA) and the Laboratory Centre for Disease Control (Canada). Clin Infect Dis 2001; 33:492503. First citation in article
7. Ochiai Y, Fukushi H, Yan C, Yamaguchi T, Hirai K. Comparative analysis of the putative amino acid sequences of chlamydial heat shock protein 60 and Escherichia coli GroEL. J Vet Med Sci 2000; 62:9415. First citation in article
8. Hammerschlag MR, Ke Z, Lu F, Roblin P, Boman J, Kalman B. Is Chlamydia pneumoniae present in brain lesions of patients with multiple sclerosis J Clin Microbiol 2000; 38:42746. First citation in article
9. Stratton CW, Sriram S. Association of Chlamydia pneumoniae with central nervous system disease. Microbes Infect 2003; 5:124953. First citation in article
10. Dong-Si T, Weber J, Liu YB, et al. Increased prevalence of and gene transcription by Chlamydia pneumoniae in cerebrospinal fluid of patients with relapsing-remitting multiple sclerosis. J Neurol 2004; 251:5427. First citation in article
11. Fainardi E, Castellazzi M, Casetta I, et al. Intrathecal production of Chlamydia pneumoniae-specific high-affinity antibodies is significantly associated to a subset of multiple sclerosis patients with progressive forms. J Neurol Sci 2004; 217:1818. First citation in article
12. Munger KL, Peeling RW, Hernan MA, et al. Infection with Chlamydia pneumoniae and risk of multiple sclerosis. Epidemiology 2003; 14:1417. First citation in article