Isolation of Chlamydia trachomatis or Ureaplasma urealyticum from the synovial fluid of patients with Reiter ' s syndrome

Background. The aim of this study was to contribute to the insight of the role of the infectious agent in ethiopathogenesis of the Reiter's syndrome development, which could directly influence the choise of treatment of these patients. Methods. Eighteen patients with urogenital form of the Reiter's syndrome and 16 controls (6 with rheumatoid arthritis and 10 with pigmented villonodular synovitis) were included in the study. In all patients standard laboratory analyses of the blood, urine and stool were made; antibody titer to Chlamydia trachomatis and Ureaplasma urealyticum was determined in synovial fluid and serum; isolation of Chlamydia trachomatis and Ureaplasma urealyticum in urethral, cervical and conjunctival swabs, as well as in prostatic and synovial fluid, was also made. HLA typing was done, too. Chlamydia was isolated in the McCoy cell culture treated with cycloheximide, while Ureaplasma was identified according to its biochemical properties grown on cellfree liquid medium. Results. Chlamydia trachomatis was isolated from the synovial fluid of 4 patients with Reiter's syndrome ( 22.2%), while Ureaplasma urealyticum was isolated in 7 of them (38.9%). These microorganisms were not found in any synovial fluid of the control group patients. Conclusion. Presence of these bacteria in the inflamed joint might be an important factor in etiopathogenesis of this disease, and it supports the hypothesis that arthritis in Reiter's syndrome is probably of the infectious origin.


Introduction
Reiter's syndrome (RS) was defined as acute reactive arthritis preceded by urogenital or gastrointestinal tract infection.Infection caused by species of several bacterial genera, including Chlamydia trachomatis (CT), Ureaplasma urealyticum (UU), Mycoplasma hominis, Yersinia, Campylobacter, Shigella, Salmonella.Giardia-Lamblia and Clostridium difficile was strongly implicated as a triggering factor for the genesis of RS, particularly in susceptible individuals positive to HLA-B27 antigen.The most recent evidence for microbial agents persistence in the inflamed joint of patients with reactive arthritis led to the assumption that RS is an infectious disease, at least at one stage of its development (1-13).
This assumption was tested in the present prospective study.The presence of bacteria in synovial fluid (SF) from patients with (CT) or (UU) triggered Reiter's syndrome was sought for.

Patients
Eighteen adult patients with Chlamydia or Ureaplasma-triggered RS diagnosed on the basis of clinical, bacteriological and serological findings were studied.Data from the findings of these patients (l female and 17 male, mean age 33) were obtained during the four-year period.A detailed history including previous sexually transmitted disease, symptoms of urethritis and a complete physical ex- amination were performed in each patient.Clinical signs of urogenital infection (urethritis, cervicitis, prostatitis) occurred in all 18 patients.Nine patients also had ocular conjunctivitis or iridocyclitis, and 11 had mucocutaneous manifestations (oral ulcer, circinate balanitis or keratoderma blennorrhagicum).As the cause of urogenital infection the bacteria (CT, UU) were examined in the swabs of the urethra or cervix, conjunctival scrapings and the prostatic fluid from all the examined patients.The patients with positive clinical or microbiologic evidence of purulent arthritis were excluded, as well as the patients with the established Yersinia, Sehigella, Salmonella, Campylobaeter and Gonoccocal reactive arthritis.Control material from 6 patients with rheumatoid arthritis (RA) diagnosed according to the American Rheumatism Association's criteria, and 10 patients with pigmented villonodular synovitis (PVNS) with affected knee joints were also studied.Physical examination, standard laboratory tests, radiography, and histopathologic examination of the synovial tissue obtained by open or arthroscopic biopsy were used as the criteria for the PVNS diagnosis.

RIA-typing
HLA type I antigenic determinants were obtained from all the patients and control subjects using standard micro-Iymphocytotoxicity assay on peripheral blood lymphocytes (14).

Synovial fluid specimens
Obtained from the knee by the open or arthroscopic surgical method synovial fluid specimens were collected into sterile heparinized tubes.Each sample of synovial fluid (SF) was divided into two parts.The first part was used for the culture on standard media for pyogens and mycobacteria.The second one, (1 ml of SF), was added to I m1 of 2SP (sucrose phosphate) transport medium, and brought immediately to the laboratory to be cultured on media specific for Ureaplasma and Chlamydia.
Ureaplasma grown on cell-free liquid medium was identified according to its biochemical properties (15), while CT was isolated by cell culture using cycloheximidetreated McCoy cells (16).

Serology
Antibodies to CT were studied by the indirect immunoperoxidase assay (Ipazyme Chlamydia: Savyon Diagnostic Ltd, Beer Sheve, Israel).The test was based on the reactivity of specific antibodies to Chlamydia inclusion bodies in infected cells fixed on masked slides.Metabolic inhibition test (International Mycolpasma, BP 70583030, Toulon Cedex 9, France) was used for antibodies to Ureaplasma.The test was based on the anti-ureaplasma antibodies inhibition not only by multiplication of the strains but also by using their enzymatic activity.
The Fischer exact test was used for statistical analysis.

Results
CT was isolated from the cervix, urethra or prostatic fluid of eleven patients and UU from nine (Table I).Both of them were isolated in 4 patients.In 3 studied patients (patients 7, 9, 11) whose urethral or prostatic-fluid cultures were negative serum antibody responses were positive to the causative microorganism: with a titre of IgG 1:64 and IgM positive specific to CT, and specific for UU with a titre ofIgG 1:16.
The results of bacteriological and serological findings in SF of patients with RS are summarized in Table 2 .
found in the control group.Difference between findings in the patients with RS and the control group was significant.According to the results the infection of urogenital tract caused by Ureaplasma semed to by more frequent trigger of the arthritis in urogenital form of RS than the one caused by Chlamydia.Cell culture for the detection of Chlamydia (synovial fluid) Four cultures (patients 1,4, 7, 13) of 18 patients with RS were positive and none in the subjects of control group.The difference was statistically significant (p<0.05).

RIA-typing
Twelve patients with RS (66.7%) were positive to B27 antigen, one with RA and none with PVNS.The compared results were statistically significant (p<O.OI).The literature contains references with positive identification of Chlamydia in synovium or SF or in both (1-8) and Ureaplasma/Pleuropneumonia-like organisms, too (8, lO, 11, 17-24).On the other hand, some authors failed to isolate these bacteria from the SF or synovial tissue samples (25,26).

Discussion
Infection by a fastidious Ureaplasma that cannot always be isolated by currently available techniques and the infection at a distant site triggering immunopathologic mechanisms is possibility difficult to exclude and will require further investigation.Ureaplasma was the cause of nongonococcal urethritis in male patients in about 60% of cases and in patients with urogenital form of Reiter's syndrome in 11.8-83.0%cases (9-11, 24, 27-29).Ureaplasma colonizes the epithelial cells of the urogenital tract exerting a direct citopathogenic effect on host cells by its toxic products and probably by activating the immune system.Urea- plasma is in fact an oportunistic bacterium and there are many hypotheses about the conditions under which it penetrates into the deep tissues becoming pathogenic.Some researches point out the importance of only some serological types of Ureaplasma, such as 8 and 4, showing a pathogenic effect (30, 31).Other experts think that host immunity disorders are necessary condition for exerting its pathogenic CrpaHa 8 BOJHOCAHI1TETCKI1 rrPErJIE,A Epoj I effect.They present 7 cases of hypogammaglobulinemia, one with congenital agammaglobulinemia, and arthritis developed in all of them.Ureaplasma was identifed in synovial fluid or synovium in all of them (32-35).The fact that Ureaplasma urogenital infection might become a systemic one was confirmed by the presence of this bacterium in bone marrow in the patients with systemic lupus erythematosus (36).According to Ginsburg and his coworkers this bacterium might be responsible for the development of .autoimmunity disorders because its chronical colonization of urogenital tract might provide constant production of small quantities of antigens (37).Thus the antigen of Ureaplasma with qualities of superantigens stimulated helper T lymphocytes specific for autoreactive B cells which developed autoimmunity by antibodies production (38).These data suggested that Ureaplasma, as weB as Chlamydia were associated with nongonococcal urethritis and might have also had an etiopathogenetic role in Reiter's syndrome.
However, the true nature of these microorganisms remains unknown.Successful chlamydial cultivation comprises the detection of infective viable elementary bodies in any given clinical sample.Regardless of the improved techniques of detection, even by using the molecular biology ones, number of positive findings of Chlamydia in the synovial fluid was not increased.One of the possible explanations of un-successful isolation may be insufficiently understood biologic features of the Chlamydia life cycle (39).For example, under certain conditions Chlamydia may enter into a phase of its life cycle during which extremely low levels of the infectious extracellular elementary bodies are produced, which is the reason for the falsely-negative culture.Besides, all unsuccessful cultivation may also be due to the presence of high titers of antichlamydial antibodies in the synovial fluid, as well as to the presence in the form of the latent organism (40).
The significance of the isolation of Ureaplasma and Chlamydia from the joint in reactive arthritis remains a subject for the debate.This is in apparent contradiction to the concept that reactive arthritis is probably an immunemediated, sterile inflammatory process which occures distantly from the primary focus of infection, mainly in genitourinary or gastrointestinal tracts (41-43).

Conclusion
It can be concluded from this study that the presence of bacteria in the inflamed joint was an important factor in the pathogenesis of Reiter's syndrome.Our findings supported the hypothesis that arthritis in RS is probably of the infectious origin.Correspondence to: Ljiljana Pavlica, Vojnomedicinska akademija, Klinika za reumatologiju i klinicku imunologiju; Cmotravska 17, 11040 Beograd, Srbija i Crna Gora.E-mail: marklena@verat.net
Chlamydia or Ureaplasma was found in synovial fluid in 61.1 % (11118) patients with the urogenital form of RS: Chlamydia was positive in 4 (22.2 %), while Ureaplasma was positive in 7 (38.9%) samples.No !,ositive results were 1

Table 1
Bacteriological and serological findings in patients with Reiter's syndrome *Cervical swab was positive for both CT and UU N.D. -not done CT -Chlamydia trachomatis UU -Ureaplasma urealyticum