Chlamydia trachomatis in reactive arthritis

Summary Evidence of deposition of chlamydial antigen in the joint was sought in 10 patients (9 of them male) with classic sexually acquired reactive arthritis, 15 women with unclassified seronegative oligoarthritis involving the knee and 15 individuals with established rheumatic disorders not associated with genital-tract or other infections. Using a fluorosceinated monoclonal antibody to the major outer membrane protein of Chlamydia trachomatis (Micro Trak, Syva) in a direct immunofluorescence test, particulate antigen with physical characteristics of chlamydial elementary bodies was seen in synovial fluid cell smears or synovial biopsies, or both, from 6, 5, and 0 patients, respectively. No typical chlamydial intracellular inclusions were seen. Corroborative evidence of recent chlamydial infection was provided by the finding of high titres of serum chlamydial antibody in all antigen-positive patients with sexually acquired reactive arthritis, including 3 from whom a genital-tract isolate was obtained, and 3 of the 5 women with unclassified arthritis. It is postulated that Chlamydia trachomatis organisms reach the joint during acute genital-tract infection, and the processing and presentation by class I major histocompatibility determinants of chlamydial antigens is a critical step in the initiation of reactive arthritis in some patients.     

Comparison of synovial tissue and synovial fluid as the source of nucleic acids for detection of Chlamydia trachomatis by polymerase chain reaction

Objective. Difficulties in detecting Chlamydia trachomatis in human joints by polymerase chain reaction (PCR) may be related to whether synovial tissue or synovial fluid (SF) is used as the source of DNA in PCR amplification. In this study, a new PCR assay was developed and used to compare chlamydial DNA in paired samples of SF and synovial tissue from patients with arthritis. Methods. The PCR assay targeted the ribosomal RNA operons, which are present in 2 copies on the C trachomatis chromosome. DNA from several relevant bacteria and chlamydial serovars was used for testing this screening system. The detection of chlamydial DNA in nucleic acid preparations from matched samples of SF and synovial tissue was compared by PCR assay. Samples were obtained from 55 patients, including patients with reactive arthritis, Reiter's syndrome, and other arthropathies. Results. Testing of the PCR screening system confirmed it to be highly specific and sensitive. Use of this assay to screen DNA from SF and synovial tissue samples showed that 29 (53%) of 55 synovial tissue preparations were positive for chlamydial DNA, but only 16 (29%) of the matched SF samples from these 29 patients were similarly positive. Five (9%) of 55 SF samples, but not their tissue counterparts, were positive for chlamydial DNA by PCR. Conclusion. Detection of chlamydial DNA in the joints of patients by PCR gives positive results more often when synovial tissue rather than SF is the source of target nucleic acids. Although synovial tissue is the source of choice for the most reliable determination of chlamydia in the joint, both synovial tissue and SF should be assayed if possible.     

SYNOVIAL FLUID AND SERUM ANTIBODIES AGAINST CHLAMYDIA IN DIFFERENT FORMS OF ARTHRITIS: INTRA-ARTICULAR IgA PRODUCTION IN CHLAMYDIA SEXUALLY ACQUIRED REACTIVE ARTHRITIS

Since the presence of Chlamydia has been shown in synovial fluid(SF) from some patients with Chlamydia reactive arthritis, weinvestigated whether anti-Chlamydia antibodies present in thejoint are derived from the circulation or are locally produced.We compared titres of IgG, IgM and IgA antibodies against Chlamydia,and against a control antigen (tetanus toxoid), by an enzyme-linkedimmunosorbent assay (ELISA), in paired samples of serum andSF from Chlamydia trachomatis sexually acquired reactive arthritis(CT-SARA) patients and from patients with other forms of arthritis.The ratio of serum/SF IgA anti-Chlamydia antibodies was significantlydecreased in CT-SARA patients. It is concluded that, in ourexperimental conditions, we found evidence for intra-articularproduction of IgA anti-Chlamydia antibodies. KEY WORDS: Chlamydia antibodies, Synovial fluid, Enzyme-linked immunoassay     

Alteration of chlamydia trachomatis biologic behavior in synovial membranes suppression of surface antigen production in reactive arthritis and reiter's syndrome

Objective. To investigate the biologic state of Chlamydia and its surface antigen expression in the synovial membranes of patients with Chlamydia-associated reactive arthritis/Reiter's syndrome (ReA/RS). Methods. Expression of chlamydial lipopoly-saccharide (LPS), major outer membrane protein (MOMP), and elementary body (EB) antigens was studied by gold labeling immunoelectron microscopy on 6 synovial membrane and 2 synovial fluid (SF) pellet samples from 6 patients with Chlamydia-associated arthritis. The study findings were compared with 24-hour cultures of HeLa cells infected with Chlamydia trachomatis EB. Results. Persistent C trachomatis infection was found in all 6 synovial membrane samples from patients who had either early or chronic arthritis. The infection persisted despite antibiotic treatment, including a 1-month course of doxycycline therapy. Most persistent organisms were atypical reticulate bodies (RBs) found in both fibroblasts and macrophages. Specific, but weak, immunogold staining for all 3 antibodies was found on both intracellular RBs and extracellular EBs. In the SF samples, Chlamydia surface antigens were detected only in phagosomes containing degraded electron-dense materials. Conclusion. The synovial membrane biopsies conducted in this study of Chlamydia-associated ReA/RS revealed atypical RBs with diminished MOMP and LPS expression. Such altered organisms may escape immune surveillance and contribute to disease chronicity; moreover, these organisms may be difficult to detect and treat in some ReA/RS patients.     

THE HUMORAL IMMUNE RESPONSE TO CHLAMYDIA TRACHOMATIS IN PATIENTS WITH ACUTE REACTIVE ARTHRITIS

Sera from 25 patients with clinical signs of reactive arthritiswere analysed for antibodies against Chlamydia trachomatis byimmunoblotting. Purified elementary bodies, purified Chlamydiaouter membrane complexes, and purified recombinant subcomponentswere used as antigens. Antibodies against C. trachomatis cysteinerich outer membrane protein 2 (Omp2) and lipopolysaccharide(LPS) were detected in 10 patients. Thus 40% of the patientspresented antibodies specific for C.trachomatis. There was nocorrelation between acute reactive arthritis and antibodiesto heat-shock proteins GroEL, GroES and DnaK. KEY WORDS: Chlamydia trachomatis, DnaK, GroEL, Omp2, Lipopolysaccharide, Humoral immune response     

Is the role of Chlamydia trachomatis underestimated in patients with suspected reactive arthritis?

Reactive arthritis is usually caused by bacteria of either the enteric or genital tracts. In the genital tract, Chlamydia trachomatis is perhaps the only aetiological agent. In Iran, newer evidence suggests that as C. trachomatis is more commonly found in the general population than was previously believed, its role in reactive arthritis may well be currently overlooked. In this review, as well as emphasizing the potential role of C. trachomatis in reactive arthritis in patients from developing countries, we also make recommendations for further clinical studies to determine its prevalence. Moreover, we also stress the need for standardization of new testing methodologies for C. trachomatis, including the use of new commercial systems in an attempt to determine a truer picture of chlamydial infection in reactive arthritis.     

Chlamydiae as etiologic agents in chronic undifferentiated spondylarthritis

Objective

The majority of patients with Chlamydia‐induced reactive arthritis do not present with the classic triad of arthritis, conjunctivitis/iritis, and urethritis. Moreover, acute chlamydial infections are often asymptomatic. The aim of the present study was to assess the prevalence of synovial Chlamydia trachomatis and Chlamydia pneumoniae infections in patients with chronic undifferentiated spondylarthritis (uSpA).

Methods

Study patients met the European Spondylarthropathy Study Group criteria for SpA, without evidence of ankylosing spondylitis, psoriasis, inflammatory bowel disease, or preceding dysentery. Symptoms were present for ≥6 months. Each patient underwent a synovial biopsy; tissue and concomitantly obtained peripheral blood mononuclear cells (PBMCs) were analyzed by polymerase chain reaction (PCR) for C trachomatis and C pneumoniae DNA. Other data collected on the day of the biopsy included standard demographic information and medical history, including any known history of C trachomatis or C pneumoniae. Physical examination (including joint count, evaluation for dactylitis and/or enthesitis, and skin examination) and HLA–B27 typing were performed. Synovial tissue (ST) samples from 167 patients with osteoarthritis (OA) were used as controls.

Results

Twenty‐six patients met the entry criteria and underwent synovial biopsy (25 knee, 1 wrist). Sixteen of them (62%) were positive for C trachomatis and/or C pneumoniae DNA (10 for C trachomatis, 4 for C pneumoniae, and 2 for both). PCR analysis of ST revealed the presence of Chlamydia significantly more frequently in patients with uSpA than in OA controls (P < 0.0001). No specific clinical characteristics differentiated Chlamydia‐positive from Chlamydia‐negative patients. PBMCs from 4 of the 26 uSpA patients (15%) were positive for Chlamydia, and Chlamydia was found in ST from 2 of these 4 patients. No significant correlation between PCR positivity and HLA–B27 positivity was found.

Conclusion

The frequency of Chlamydia‐positive ST samples, as determined by PCR, was found to be significantly higher in patients with uSpA than in patients with OA. Our results suggest that in many patients with uSpA, chlamydial infection, which is often occult, may be the cause.

     

Yersinia–triggered reactive arthritis. use of polymerase chain reaction and immunocytochemical staining in the detection of bacterial components from synovial specimens

Objective. To investigate whether microbial DNA is present in synovial specimens from patients with Yersinia-triggered reactive arthritis. Methods. Synovial specimens from 13 patients with Yersinia enterocolitica O:3-triggered reactive arthritis and from 16 control patients were studied using polymerase chain reaction and immunocytochemical staining techniques. Results. Yersinia chromosomal DNA was not found in any of the synovial specimens from Yersinia-triggered arthritis patients or controls, whereas with immunocytochemical techniques, Yersinia antigens were observed in synovial specimens from all of the patients with Yersinia-triggered reactive arthritis. Conclusion. Only stable bacterial degradation products, not whole bacteria, are present at the site of inflammation in Yersinia-triggered reactive arthritis.     

THE VALUE OF ISOTYPE DETERMINATION OF SERUM ANTIBODIES AGAINST CHLAMYDIA FOR THE DIAGNOSIS OF CHLAMYDIA REACTIVE ARTHRITIS

In clinical rheumatology, the diagnosis of Chlamydia reactivearthritis is difficult because an incomplete form of the diseasecan closely resemble an undifferentiated seronegative mono/oligoarthritis.We investigated whether measuring specific isotypes of anti-Chlamydiaantibodies in serum can improve the diagnosis, by comparingsuch antibody concentrations in the serum of patients with well-defineddisease, i.e. Chlamydia trachomatis sexually acquired reactivearthritis (CT-SARA), with other arthritides. Antibody levelswere determined by enzyme-linked immunosorbent assay (ELISA).When considering two different isotypes and their combination,the best sensitivity (63%) was obtained for IgM and/or IgA resultswith a specificity of 81%. The patients with CT-SARA and SARAhad the highest levels of antibodies of all isotypes tested.It is concluded that, in our experimental conditions, only veryhigh values of specific isotypes could indicate a diagnosisof Chlamydia reactive arthritis. KEY WORDS: Sexually acquired reactive arthritis, Chlamydia, Antibodies, Enzyme-linked immunoassay     

Chlamydial rRNA in the joints of patients with Chlamydia-induced arthritis and undifferentiated arthritis.

Synovial fluid and synovial membrane specimens of 11 patients with Chlamydia-induced arthritis (CIA), 24 patients with undifferentiated arthritis (UndA), 4 patients with post-enteritic reactive arthritis, 3 patients with Lyme arthritis and 9 patients with rheumatoid arthritis were investigated for the presence of Chlamydia trachomatis (C. trachomatis). A single stranded DNA-probe was used for nucleic acid hybridization with ribosomal RNA (rRNA) from C. trachomatis. In 4 patients (CIA = 1, UndA = 3) chlamydial rRNA was found in the synovial fluid. In one additional patient (CIA) the specimen of a synovial membrane biopsy was positive for chlamydial rRNA. The detection of intra-articular chlamydial rRNA is discussed as an indicator for the presence of viable Chlamydiae in inflamed joints.     

Yersinia-triggered reactive arthritis. Use of polymerase chain reaction and immunocytochemical staining in the detection of bacterial components from synovial specimens.

OBJECTIVE. To investigate whether microbial DNA is present in synovial specimens from patients with Yersinia-triggered reactive arthritis. METHODS. Synovial specimens from 13 patients with Yersinia enterocolitica O:3-triggered reactive arthritis and from 16 control patients were studied using polymerase chain reaction and immunocytochemical staining techniques. RESULTS. Yersinia chromosomal DNA was not found in any of the synovial specimens from Yersinia-triggered arthritis patients or controls, whereas with immunocytochemical techniques, Yersinia antigens were observed in synovial specimens from all of the patients with Yersinia-triggered reactive arthritis. CONCLUSION. Only stable bacterial degradation products, not whole bacteria, are present at the site of inflammation in Yersinia-triggered reactive arthritis.     

THE POSSIBLE ROLE OF SHIGELLA IN SPORADIC ENTERIC REACTIVE ARTHRITIS

Reactive arthritis (ReA) occurs after a urogenital infectionusually with Chlamydia trachomatis or an enteritis due to Yer-sinia,Salmonella, Campylobacter or Shigella, Shigella, except duringepidemics, is not considered to be a frequent cause of entericreactive arthritis. However this might be due to the lack ofa reliable antibody test, which makes diagnosis difficult. Wecompared synovial and peripheral blood lymphocyte proliferationto various bacterial antigens in 19 consecutive patients withReA or undifferentiated oligoarthritis. In five patients Shigellawas identified as the causative microbe by a specific synoviallymphocyte proliferation. All five patients had a history ofsymptomatic diarrhoea and had negative stool cultures by thetime arthritis developed. Four of the five were HLA B 27 positive.We conclude that Shigella may be underestimated as a cause ofnon-epidemic ReA. KEY WORDS: Reactive arthritis, Shigella, Antigen specific lymphocytes, Synovial fluid     

Chlamydial infection preceding the development of rheumatoid arthritis: a brief report

Chlamydia trachomatis-triggered reactive arthritis is a well-documented entity that has been extensively described. We do not have a clear understanding about the inflammatory oligoarthritis associated with the presence of this organism. It is rarely cultured from the synovial fluid, but is usually detectable by molecular biological techniques. Typically, Chlamydia trachomatis causes a sterile but inflammatory oligoarthritis. We report an unusual case of inflammatory monoarthritis in a young woman in whom Chlamydia was isolated from the synovial fluid. This is the first case of documented isolation of Chlamydia from synovial fluid, which subseqently was diagnosed as rheumatoid arthritis.     

CHLAMYDIAL DNA IS ABSENT FROM THE JOINTS OF PATIENTS WITH SEXUALLY ACQUIRED REACTIVE ARTHRITIS

The polymerase chain reaction was used to detect the presenceof a plasmid essential for the growth of Chlamydia tracho-matis.As few as 10 copies of the plasmid in the initial reaction mixwere detectable using this technique. In contrast, chla-mydialDNA was not detectable in the knee joints of nine patients withdefinite sexually acquired reactive arthritis (SARA) or ninepatients with suspected SARA. Five patients with an undifferentiatedseronegative lower limb oli-goarthropathy, one with Crohn'sdisease and another with post-enteric reactive arthritis hadevidence of intra-articular chlamydial antigens as judged byfluorescein-labelled monoclonal antibody staining of joint materialbut, again, no chlamydial plasmid DNA was detected. The natureof the immunofluorescent staining seen in some of these samplesremains to be elucidated. It could be due to the presence ofchlamydial outer membrane protein or lipopolysaccharide antigensin the joints, either free or in immune complexes, or it maybe artefactual. Our results indicate that viable C. trachomatisis not present in the joints of the patients in this study evenin the presence of chlamydial antigen detected by fluorescentantibody testing KEY WORDS: Polymerase chain reaction, Joint disease     

Light and electron microscopic studies on the synovial membrane in reiter's syndrome.

Studies by light microscopy on synovium obtained from 11 patients with Reiter's syndrome during the first month of an episode showed proliferation of synovial lining cells, polymorphonuclear neutrophils among the synovial lining cells, increased surface fibrin, and vascular congestion. Biopsy specimens taken later showed vascular congestion and still proliferated synovial lining cells, fewer polymorphonuclear neutrophils in some, and a tendency toward increased infiltration with lymphocytes and plasma cells. Electron microscopy of samples from 8 patients during the first month of disease activity showed occlusion of vessels by platelets in 4, and fibrin or dense granular material in the vessel walls in 4. Five of the patients with arthritis of less than 4 weeks duration had unidentified intracellular and extracellular particles; some of these were highly suggestive of Chlamydia. No such particles were noted in samples from patients with more chronic cases. Using an antibody to Chlamydia trachomatis and the peroxidase-antiperoxidase technique, immunocytochemistry showed reaction product in synovial macrophages in 2 patients with arthritis of less than 4 weeks duration, but not in the 1 patient studied who had more chronic disease. These studies provide support for dramatic synovial vascular injury consistent with that caused by endotoxin and the presence of chlamydial antigen in synovial macrophages, at least in the early phases of synovitis.     

Chlamydia and Borrelia DNA in synovial fluid of patients with early undifferentiated oligoarthritis: Results of a prospective study

Objective

More than 50% of patients with synovitis involving 1–4 joints remain classified as having undifferentiated oligoarthritis (UOA) after 1 year of disease. The clinical presentation is often similar to that of reactive arthritis (ReA) and other spondylarthropathies or to Lyme arthritis. We therefore determined how often Chlamydia trachomatis (Ct) and Borrelia burgdorferi (Bb) can be identified in patients with UOA, by using an extensive laboratory approach.

Methods

We prospectively studied 52 patients with UOA who presented at an early synovitis clinic in a region highly endemic for Lyme disease. Patients were examined by standardized clinical and immunoserologic procedures. Synovial fluid was screened for the presence of Ct and Bb DNA by polymerase chain reaction (PCR). Urine was tested for Ct DNA by ligase chain reaction, and serum was tested for Ct antibodies by enzyme‐linked immunosorbent assay and Bb antibodies by hemagglutination test and Western blotting. PCR results in the UOA patients were compared with the results in cohorts of patients with definite rheumatoid arthritis (RA), Lyme arthritis, and Chlamydia‐induced arthritis (CIA).

Results

In the synovial fluid of 9 of 52 patients with UOA (17%), we found Ct DNA, and in 6 of the 52 patients (12%), Bb DNA was found. The frequency of bacteria‐specific DNA was 50% (7 of 14) in CIA patients and 69% (11 of 16) in patients with Lyme arthritis. No Bb or Ct DNA was found in the synovial fluid of the 31 RA patients.

Conclusion

With optimized PCR protocols, it is possible to detect considerable levels of Bb and Ct DNA in the synovial fluid of patients with UOA. Although the presence of bacterial DNA does not unequivocally prove its etiologic significance, we suggest that at least one‐third of patients with UOA may have a form of ReA that involves asymptomatic primary infection.

     

Evaluation of amplicor chlamydia PCR and LCX chlamydia LCR to detect Chlamydia trachomatis in synovial fluid

OBJECTIVES: PCR has been successfully used in research for the detection of C. trachomatis DNA in synovial samples. However, each research laboratory has developed its own PCR, making inter-laboratory comparisons difficult. To allow for standardization we evaluated two commercially available amplification systems originally designed for the examination of urogenital samples (Roche Amplicor Chlamydia PCR and Abbott LCX Chlamydia LCR), using them to analyse spiked and clinical synovial fluid (SF) samples from reactive arthritis (ReA), undifferentiated arthritis (UA), and rheumatoid arthritis (RA) patients. We compared their sensitivity in assays of clinical SF samples with our in-house developed C. trachomatis specific nested PCR. METHODS: SF was spiked with purified C. trachomatis elementary bodies (EB) and analyzed by the commercial assays. Clinical SF samplesfrom ReA (n=21), UA (n=79) and RA (n=50) patients were examined by the two commercial assays and our in-house PCR. RESULTS: Using SF samples spiked with defined numbers of C. trachomatis EB, the sensitivity of the commercial tests was high and similar to published PCR sensitivity. In clinical SF specimens the commercial assays was also able to detect CT; however, the in-house PCR was more sensitive. Out of 10 PCR-positive SF samples Amplicor tested positive in only 4/10 and LCX in only 3/10. The in-house PCR detected chlamydial DNA in synovialfluidfrom 5/21 ReA (24%), 5/79 UA (6%) and in none of the 50 RA patients. CONCLUSION: Commercial amplification assays allow the detection of C. trachomatis in clinical specimens, although with a lower sensitivity than optimized PCR. Potential explanations are discussed.     

Molecular detection of bacterial DNA in venereal-associated arthritis

Objective. To evaluate the utility of polymerase chain reaction (PCR) amplification in detecting DNA from venereal-associated microorganisms in the synovial fluid of patients with inflammatory arthritis. Methods. Oligonucleotide primers were developed for nested PCR based on Chlamydia, Ureaplasma, and Neisseria DNA sequences. PCR products were detected by gel electrophoresis and dot-blot hybridization. Primers specific for the target bacterial DNA were used to search for bacterial DNA in 61 synovial fluid specimens from patients with inflammatory arthritis, including several clinically associated with venereal infection. Results. Five of the 61 synovial fluid specimens were positive for Neisseria gonorrhoeae DNA. Four of the 5 patients had clinical diagnoses of gonococcal arthritis; the other patient had an unexplained monarthritis. One specimen from a patient with a clinical diagnosis of gonococcal arthritis was negative for N gonorrhoeae. Three of the 61 specimens were positive for Chlamydia DNA. Two were derived from patients with clinical diagnoses of reactive arthritis or Reiter's syndrome, and 1 was from a patient with unexplained monarthritis. One of the 61 specimens was positive for Ureaplasma DNA; this sample was from a patient with a clinical diagnosis of Reiter's syndrome. In an additional patient with Reiter's syndrome, Ureaplasma DNA was also found in prostate biopsy tissue and a urine sample obtained after prostate massage (synovial fluid not available). Conclusion. These data support the classification of these 3 venereal-associated arthritides as infectious processes, and suggest that PCR for bacterial DNA is a useful method for detecting infectious agents in synovial fluid.     

Early cytokine profiles in the joint define pathogen clearance and severity of arthritis in Chlamydia‐induced arthritis in rats

Objective

Although Chlamydia trachomatis–induced arthritis is among the most common rheumatic diseases having an identified infectious trigger, the pathogenesis of this arthritis is not well defined. We sought to investigate the host–microbe interactions that contribute to the severity of arthritis initiated by chlamydial infection.

Methods

We established an experimental rat model of C trachomatis–induced arthritis that recapitulates many pathologic features of the clinical disease. The severity of the arthritis was defined using an established histopathologic scoring system. Host clearance of the pathogen and local cytokine production were examined by enzyme‐linked immunosorbent assays.

Results

Lewis rats were susceptible to C trachomatis–induced arthritis, whereas BN rats were relatively resistant to this disease. Significant differences in the histopathologic severity of arthritis were originally observed on day 21, and this prompted an examination of the acute phase of the arthritis. As early as day 5 after the onset of the arthritis, pathologic changes in Lewis rats were more severe than those in BN rats. An evaluation of the role of complement using cobra venom factor treatment excluded complement as being the key to differential sensitivity, because decomplementation did not eliminate the differences in arthritis severity between Lewis and BN rats. Host clearance, in contrast, was significantly different between the rat strains, with BN rats showing more prompt and effective clearance of the pathogen from both synovial tissues and spleen compared with Lewis rats. Local cytokine profiles demonstrated that host resistance was characterized by enhanced synovial expression of tumor necrosis factor α, interferon‐γ (IFNγ), and interleukin‐4.

Conclusion

These studies demonstrated that cytokines thought to be proinflammatory in nature can play an important role in host defense in infection‐triggered arthritis and serve to highlight the dynamic cytokine relationships that constitute effective host–pathogen interactions.