LOWER PREVALENCE OF CHLAMYDIA PNEUMONIAE DNA COMPARED WITH CHLAMYDIA TRACHOMATIS DNA IN SYNOVIAL TISSUE OF ARTHRITIS PATIENTS

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1 ARTHRITIS & RHEUMATISM Vol. 42, No. 9, September 1999, pp ,American College of Rheumatology 1889 LOWER PREVALENCE OF CHLAMYDIA PNEUMONIAE DNA COMPARED WITH CHLAMYDIA TRACHOMATIS DNA IN SYNOVIAL TISSUE OF ARTHRITIS PATIENTS H. RALPH SCHUMACHER, JR., HERVÉ C. GÉRARD, THURAYYA K. ARAYSSI, JOSÉ A. PANDO, PATRICK J. BRANIGAN, DIEGO L. SAAIBI, and ALAN P. HUDSON Objective. To assess the presence of Chlamydia pneumoniae DNA in the joints of patients with reactive arthritis (ReA) and other arthritides. Methods. DNA was prepared from synovial tissue (ST) and several synovial fluid (SF) samples from 188 patients with either ReA, undifferentiated oligoarthritis, or other forms of arthritis, and from 24 normal (nonarthritis) individuals. Preparations were screened using polymerase chain reaction (PCR) assays that independently targeted the C pneumoniae 16S ribosomal RNA and major outer membrane protein genes. Results. Twenty-seven of 212 ST samples (12.7%) were PCR positive for C pneumoniae DNA; 10 SF samples from these 27 patients were similarly positive. Among the PCR-positive patients, 3 had ReA, 2 had Reiter s syndrome, 7 had undifferentiated oligoarthritis, 4 had undifferentiated monarthritis, 6 had rheumatoid Supported by grants from the Arthritis Foundation of Eastern Pennsylvania, the Department of Veterans Affairs Medical Research Service, the NIH (grant no. AR-42541), the NIH Clinical Research Center at the Hospital of the University of Pennsylvania, and an Intergovernmental Personnel Act agreement between the University of Pennsylvania and the Arthritis and Rheumatism Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, NIH. H. Ralph Schumacher, Jr., MD: University of Pennsylvania School of Medicine and Department of Veterans Affairs Medical Center, Philadelphia, Pennsylvania, and National Institute of Arthritis and Musculoskeletal and Skin Diseases, NIH, Bethesda, MD; Hervé C. Gérard, PhD, Alan P. Hudson, PhD: Wayne State University School of Medicine, Detroit, Michigan; Thurayya K. Arayssi, MD (current address: American University of Beirut Medical Center, Beirut, Lebanon), José A. Pando, MD: National Institute of Arthritis and Musculoskeletal and Skin Diseases, NIH, Bethesda, Maryland; Patrick J. Branigan, BS, Diego L. Saaibi, MD: University of Pennsylvania School of Medicine, Philadelphia. Drs. Schumacher and Hudson contributed equally to this work. Address reprint requests to H. Ralph Schumacher, Jr., MD, Medical Research, Department of Veterans Affairs Medical Center, University and Woodland Avenues, Philadelphia, PA Submitted for publication October 1, 1998; accepted in revised form April 22, arthritis, and 5 had other forms of arthritis. No samples from normal control individuals were PCR positive. Conclusion. DNA of C pneumoniae is present in synovial specimens from some arthritis patients. The prevalence of this organism in the joints was lower than that of C trachomatis, and synovial presence of the organism was not associated with any distinct clinical syndrome. Widely disseminated nucleic acids such as those of C pneumoniae might have some role in the pathogenesis of several arthritides, since the organism was not found in the ST from normal control individuals. Chlamydia pneumoniae is a bacterial pathogen whose primary sites of infection are the respiratory and oral mucosa (1). This organism is a significant etiologic agent in pneumonia and other respiratory conditions (2), and recent studies have associated C pneumoniae infection with unexpected clinical manifestations, including atherosclerosis (3). Epidemiologic studies have indicated that the overall prevalence of infection with C pneumoniae is higher than that of Chlamydia trachomatis in virtually all populations examined. Furthermore, studies suggest that virtually everyone is infected with the organism at some time during his or her lifetime, and that reinfection may be common (4). Several bacterial species have been associated with the pathogenetic process ending in reactive arthritis (ReA) and its subset Reiter s syndrome (RS), including Campylobacter, Yersinia, and Chlamydia (5). Among bacteria associated with ReA and RS, however, C trachomatis has emerged as a prominent agent, largely because of its high prevalence in the population (5). Many previous polymerase chain reaction (PCR) based studies have demonstrated that DNA from this organism is often present in synovial tissue (ST) of patients with ReA/RS (for example, see ref. 6), and we recently

2 1890 SCHUMACHER ET AL showed that the bacterium is viable and metabolically active during its residence in synovium (7). Some studies have raised the possibility that C pneumoniae, similar to C trachomatis, may be a causative agent in ReA (8 10). This suggestion was based initially on serologic data and lymphocyte proliferation assays, rather than on direct demonstration of the organism or its molecular components in synovial material. Detection in the synovium has become an essential aspect of establishing both an etiologic relationship between an infecting organism and ReA, and in confirming the diagnosis. One group has reported identification of C pneumoniae DNA in 1 of 55 synovial fluid (SF) samples using a PCR screening system (11); this is a far lower prevalence than that shown in previous studies that targeted C trachomatis in patients with ReA/RS (6). Nonetheless, the high prevalence of infection with C pneumoniae, combined with serologic and other indirect evidence, as well as its close relationship with C trachomatis, all have suggested that infection with this organism might be involved in the pathogenesis of ReA/RS. Culture of C pneumoniae is difficult, and therefore, molecular evidence that the organism or its molecular components are found in synovia of affected individuals would be important. We undertook studies to determine whether C pneumoniae DNA could be found in the ST and/or SF from a large group of patients with various forms of arthritis. Using well-characterized PCR assays, we demonstrated that DNA from C pneumoniae can be identified in ST from 13% of patients examined. PATIENTS AND METHODS Patients and samples. DNA samples originated from 188 patients who were attending the Rheumatology Clinics at the Hospital of the University of Pennsylvania and the Department of Veterans Affairs Medical Center in Philadelphia, and the Early Arthritis Research Program at the National Institutes of Health (NIH) in Bethesda. When originally procured, synovial biopsy specimens, which were obtained with a Parker- Pearson needle, were immediately snap-frozen in liquid nitrogen and stored at 70 C. Some SF samples were procured from the same patients by arthrocentesis prior to biopsy. All samples were number-coded prior to assay. In addition to arthritis patients, 24 normal controls from an approved research program provided tissue via needle synovial biopsy as part of the NIH Early Arthritis Study Program. Patient samples included in this study were chosen essentially randomly in terms of diagnosis, from the archives of our patient population. Samples were, however, selected on the basis of having enough DNA of adequate quality for the analyses intended. Patient diagnoses included ReA, RS, rheumatoid arthritis (RA), osteoarthritis, and other arthritides. Diagnoses were made according to the American College of Rheumatology criteria (12). Some patients had a history of respiratory illness just preceding the onset of arthritis; all presumably had upper respiratory illness at some time. Patients were diagnosed with undifferentiated arthritis if they did not meet the criteria for RA, but had inflammatory arthritis without features of antecedent infection with agents known to trigger ReA. Nucleic acid preparation and analysis. Nucleic acids were prepared from ST and SF pellets as previously described (6,7). Elementary bodies (EB) of C pneumoniae strain TW-183 were obtained from American Type Culture Collection (Rockville, MD). The EB of C trachomatis serovar C were a gift of Dr. J. A. Whittum-Hudson (Johns Hopkins University, Baltimore, MD). DNA was prepared from the EB of both species for control amplifications as previously described (6,7). Assays to screen for C pneumoniae DNA targeted the 16S ribosomal RNA (rrna) gene and the major outer membrane protein gene OmpA, each using published primer systems (13,14). Amplification products were analyzed on agarose gels and confirmed by hybridization. All assays were performed in duplicate and conducted independently by 2 different investigators (HCG and PJB) in a blinded manner. Samples were considered positive for C pneumoniae DNA only when the results of both PCR assays were positive. All preparations were also assessed by PCR for C trachomatis DNA in independent assays, as previously described (6,7). RESULTS PCR analyses. Figure 1 provides representative examples of typical PCR screening results for a patient ST sample that was negative in both the C pneumoniae and C trachomatis PCR assay systems (lanes 2 and 3), a sample that was positive for C pneumoniae DNA but negative for C trachomatis DNA (lanes 4 7), and a sample that was positive for DNA of C trachomatis (lanes 8 11). Positive samples usually gave strong amplification signals, and negative samples were unequivocally so. Table 1 presents a general summary of patient characteristics and diagnoses for the study population, as well as a summary of PCR screening results. Amplifications of DNA from ST samples were either negative in both the 16S rrna and OmpA-directed assays or positive in both. Of the 212 tissue samples analyzed, 12.7% were PCR positive for C pneumoniae DNA, 28.8% for C trachomatis DNA, and 2.4% for both. In patients with ReA, 7.9% of synovial biopsy samples were PCR positive for C pneumoniae, but 52.6% were PCR positive for C trachomatis, whereas 5.3% were positive for both organisms. Similarly, 11.8% of RS patient samples were PCR positive for C pneumoniae, 76.4% for C trachomatis, and 5.9% for both bacteria. Importantly, no samples from normal (nonarthritis) control individ-

3 C PNEUMONIAE AND ARTHRITIS 1891 Figure 1. Representative polymerase chain reaction (PCR) screening results in patient samples using the Chlamydia pneumoniae 16S ribosomal RNA (rrna) and outer membrane protein A (OmpA) directed primer sets. Nucleic acids were prepared from patient samples, HeLa cells, C pneumoniae elementary bodies (EB), and Chlamydia trachomatis EB (as described in Patients and Methods). Lane 1, 100-bp size standards; lanes 2 and 3, 16S rrna and OmpA directed PCR assays, respectively, using, as template, DNA from a synovial tissue sample negative for C pneumoniae in each assay; lanes 4 and 5, 16S rrna and OmpA directed PCR assays, respectively, using, as template, nucleic acids from a synovial tissue sample positive for C pneumoniae in each assay; lanes 6 and 7, PCR assays targeting the C trachomatis 16S rrna and Omp1 genes, respectively, using nucleic acids from the same patient sample as assessed in lanes 4 and 5, and using primers specific for those genes; lanes 8 and 9, 16S rrna and OmpA directed PCR assays, respectively, using nucleic acids from a synovial sample positive for C trachomatis in each assay; lanes 10 and 11, PCR assays targeting the C trachomatis 16S rrna and Omp1 genes, respectively, using nucleic acids from the same patient sample as assessed in lanes 8 and 9, and using primers specific for those genes. uals were PCR positive for C pneumoniae, although 2 were positive for C trachomatis. The percentage of C pneumoniae positive patients with undifferentiated oligoarthritis was lower than that of patients with ReA/RS (Table 1); similarly, a lower percentage of patients with undifferentiated oligoarthritis was positive for C trachomatis by PCR. Among the small number of patients studied who had undifferentiated monarthritis, 4 of 5 were PCR positive for C pneumoniae DNA, but none were positive for C trachomatis. Interestingly, several RA patient samples were PCR positive for C pneumoniae or C trachomatis, while 2 were PCR positive for both. Among the SF samples studied, 9 were PCR positive for C pneumoniae DNA (1 with RS, 1 with ReA, 2 with RA, 3 with undifferentiated oligoarthritis, 1 with undifferentiated monarthritis, and 1 other). Thus, the prevalence of C pneumoniae DNA in ST was lower than that of C trachomatis DNA, and that prevalence was lower in virtually every patient group. Among diagnostic groups for which relatively large numbers of patients were assayed (ReA, RA, and undifferentiated oligoarthritis), the percentage of patients who were positive for the C pneumoniae organism by PCR did not differ significantly. Analysis of patients PCR positive for C pneumoniae DNA. We performed a thorough analysis of the clinical information for each patient whose synovial biopsy specimen was PCR positive for C pneumoniae, but we could identify no set of characteristics specific to those individuals. About one-third of these PCRpositive patients were female (9 of 27), and within each diagnostic group, there appeared to be no sex bias in the distribution of such positivity. Ages for PCR-positive individuals ranged relatively evenly from 19 years to 75 Table 1. Working diagnosis General characteristics of the patient population studied* Number studied Number F/M Mean age/range, years Average disease duration/range, months Number (%) PCR positive Cp Ct Cp Ct ReA 38 18/ / / (7.9) 20 (52.6) 2 (5.3) RS 17 4/ / / (11.8) 13 (76.5) 1 (5.9) RA 43 32/ / / (14.0) 13 (30.2) 2 (4.7) UO 80 45/ / / (8.8) 13 (16.3) 0 Umono 5 1/4 42.0/ / (80) 0 0 Normal 24 15/9 39.9/23 65 NA 0 2 (8.3) 0 Other 5 0/5 60.0/ / (100) 0 0 Total /97 27 (12.7) 61 (28.8) 5 (2.4) * PCR polymerase chain reaction; Cp Chlamydia pneumoniae; Ct Chlamydia trachomatis; ReA reactive arthritis; RS Reiter s syndrome; RA rheumatoid arthritis; UO undifferentiated oligoarthritis; Umono undifferentiated monarthritis; NA not applicable. Controls with no arthritis. Includes patients with osteoarthritis, septic arthritis, Lyme disease, psoriatic arthritis, and Behçet s disease.

4 1892 SCHUMACHER ET AL years, and disease durations ranged from 0.01 years to 32 years. Many patients having C pneumoniae DNA had a history of infection, including recent respiratory illness, and some had a history of antibiotic use; we found no correlation between PCR positivity for C pneumoniae and previous use or non-use of antibiotics. Histologic examination of ST samples from PCR-positive patients showed no characteristics unique to those samples (data not shown). We could identify no relationship between the presence of bacterial DNA in the joint and HLA B genotype, but typing results were not available for all patients. DISCUSSION Studies from other groups have suggested indirectly that C pneumoniae, a newly described chlamydial species, may be involved in pathogenesis leading to ReA in some patients (8 10). To determine whether nucleic acids from this organism are present in synovia, we used PCR assays to assess whether chromosomal DNA from C pneumoniae could be identified in the joints of patients with ReA and/or other forms of arthritis. Of 212 ST samples screened, 13% were PCR positive for the organism; only 4% of the limited number of SF samples from these patients were similarly positive. The PCR-positivity rate for C pneumoniae in ST did not appear to be much different among the 3 diagnostic groups for which we had relatively large sample numbers (i.e., ReA, RA, and undifferentiated oligoarthritis; 38 patients/group), in which the rate ranged from 8% to 14%. Of ST assayed from patients with RS, 12% were PCR positive for the organism. The identification of DNA from C pneumoniae in 4 of 5 patients with undifferentiated monarthritis is of interest, but the number studied is too low for any conclusions to be drawn. Importantly, no ST samples from control individuals were positive for C pneumoniae. The patient samples examined were derived from a large, ongoing study of early ReA and other arthritis, and the samples represent a relatively selected study population in terms of diagnosis. Indeed, 25% of patients from whom samples were procured had diagnoses of ReA or RS, and many others had other arthritides initially selected for study because they were thought to be potentially attributable to infectious causes; many of these remain classified as undifferentiated. It is of interest that of the samples assayed from ReA/RS patients, 9% were PCR positive for C pneumoniae, while 60% were PCR positive for C trachomatis DNA. In the ST samples from the large group of patients with undifferentiated oligoarthritis, the rates of PCR positivity for C pneumoniae and C trachomatis were nearly 2-fold different ( 9% versus 16%, respectively). Given the information available, we could identify neither specific nor general patient characteristics that were convincingly associated with the presence of C pneumoniae in synovial materials. Moreover, there were no clear differentiating characteristics in the patients who were PCR positive for the organism in ST versus SF. In those patients for whom anti C pneumoniae antibodies were assessed, the percentage of those who were PCR positive for C pneumoniae did not differ significantly from the percentage of those who were PCR negative for the organism. Moreover, the proportion of HLA B27 positive patients in the C pneumoniae positive group was about the same as that in the overall patient population studied. Nonetheless, although the synovial presence of C pneumoniae DNA occurred at a lower rate than that of its sister species in all diagnostic groups studied, that rate was higher than seen in our normal control samples (0 of 24), suggesting that further study of some possible relationship between C pneumoniae infection and joint disease is warranted. The results presented demonstrate that intact DNA of C pneumoniae can be identified in ST, and more rarely in SF, from patients with various diagnoses. The latter finding is reasonably consistent with the results of a recent PCR-based examination by another group that assessed only SF samples from 55 patients (11) and found that only a single sample proved to be PCR positive for the organism. Moreover, our observation that chlamydial DNA is far more prevalent in the ST from arthritis patients than it is in the SF from the same individuals is consistent with the results of a study from this group targeting nucleic acids from C trachomatis (6). However, whether C pneumoniae is a causal factor in the disease pathogenesis in any of the patients studied remains to be determined. In this context, we noted that ST samples from 5 patients who were PCR positive for C pneumoniae DNA in our assays were also PCR positive for C trachomatis DNA in independent assays. Thus, it is not clear which, if either, is the causative agent for disease in these cases. There were no clear differentiating clinical or other features in the patients who had DNA from both organisms in ST, as opposed to those singly infected with either organism at that site. Further studies will be needed to examine any implications of this evidence for dissemination of C pneumoniae to the joints. In addition, the surprisingly high incidence of C trachomatis in RA joints suggests that host response and other features must be examined

5 C PNEUMONIAE AND ARTHRITIS 1893 along with any PCR findings to determine the significance of bacterial nucleic acids. Further characterization of patient samples by analyses targeting DNA from other infectious organisms, and studies of individual genetic backgrounds and predispositions, may provide insight into the biologic effects of articular bacterial products. ACKNOWLEDGMENTS We are grateful to collaborators at the Hospital of the University of Pennsylvania, the Department of Veterans Affairs Medical Center in Philadelphia, and the National Institute of Arthritis and Musculoskeletal and Skin Diseases, who evaluated and followed up the study patients; these colleagues include John Klippel, MD, Percio Gulko, MD, Hani El Gabalawy, MD, Rafaela Goldbach-Mansky, MD, Cheryl Yarboro, RN, and Marianna Crane, RN. REFERENCES 1. Grayston JT. Chlamydia pneumoniae, strain TWAR pneumonia. Annu Rev Med 1992;43: Grayston JT, Aldous MB, Easton A. Evidence that Chlamydia pneumoniae causes pneumonia and bronchitis. J Infect Dis 1993; 168: Campbell LA, O Brien ER, Cappuccio AL, Kuo CC, Wang SP, Stewart D, et al. Detection of Chlamydia pneumoniae TWAR in human coronary atherectomy tissues. J Infect Dis 1995;172: Leinonen M. Pathogenetic mechanisms and epidemiology of Chlamydia pneumoniae. Eur Heart J 1993;14: Köhler L, Zeidler H, Hudson AP. Etiologic agents in reactive arthritis, their molecular biology and phagocyte-host interactions. Baillieres Clin Rheumatol 1998;12: Branigan PJ, Gérard HC, Hudson AP, Schumacher HR Jr. Comparison of synovial tissue and synovial fluid as source of nucleic acids for detection of Chlamydia trachomatis by polymerase chain reaction. Arthritis Rheum 1996;39: Gérard HC, Branigan PJ, Schumacher HR, Hudson AP. Synovial Chlamydia trachomatis in patients with reactive arthritis/reiter s syndrome are viable but show aberrant gene expression. J Rheumatol 1998;25: Gran JT, Hjetland R, Andraessen AH. Pneumonia, myocarditis and reactive arthritis due to Chlamydia pneumoniae. Scand J Rheumatol 1993;22: Braun J, Laitko S, Treharne J, Eggens U, Wu P, Distler A, et al. Chlamydia pneumoniae a new causative agent of reactive arthritis and undifferentiated oligoarthritis. Ann Rheum Dis 1994;53: Beaudreuil J, Hayem G, Meyer O, Khan MF. Reactive arthritis ascribed to Chlamydia pneumoniae. Rev Rhum 1995;3: Wilkinson NZ, Kingsley GH, Sieper J, Braun J, Ward ME. Lack of correlation between the detection of Chlamydia trachomatis DNA in synovial fluid from patients with a range of rheumatic diseases and the presence of an antichlamydial immune response. Arthritis Rheum 1998;41: Schumacher HR Jr, Klippel JN, Koopman WJ, editors. Primer on the rheumatic diseases. 10th ed. Atlanta: Arthritis Foundation; Gaydos CA, Quinn TC, Eiden JJ. Identification of Chlamydia pneumoniae by DNA amplification of the 16S rrna gene. J Clin Microbiol 1992;30: Balin BJ, Gérard HC, Arking EJ, Appelt DM, Branigan PJ, Abrams JT, et al. Identification and localization of Chlamydia pneumoniae in the Alzheimer s brain. Med Microbiol Immunol 1998;187:23 42.