The Relation between Chronic Aseptic Arthritis and Previous Chlamydia Infection among Middle Aged Iraqi Patients

Aims: This study was conducted to detect and evaluate the relation between chronic aseptic arthritis and previous Chlamydia trachomatis infection among middle aged Iraqi patients. Study Design: Cross sectional study Methodology: This study included 33 known patients with chronic arthritis, with negative rheumatoid factor (RF) in their sera, aging from 26 to 61 years. Septic arthritis was excluded by history, clinical examination, and blood investigations. The ESR for these patients was estimated. Other 33 subjects were taken as a control group. Chlamydia trachomatis IgM and IgG were estimated by ELISA test (sandwich method) for both groups' sera, and results were statistically Clinical Practice Article analyzed. Results: The female: male ratio in current study is (5.6: 1), with mean age of (46) years old. More than half of cases (52%) were having ESR value of 40-60. A highly significant statistical difference ( P <0.001) was noticed between study groups in regard to C. trachomatis IgG mean levels in their sera calculated by unpaired T- test. Conclusion: There is a significant relation between chronic arthritis and previous Chlamydia infection in current study (demonstrated by high titer of anti- Chlamydia trachomatis IgG).


INTRODUCTION
Aseptic arthritis is precipitated by an extraarticular infection. The disease has attracted a great deal of research interest because it is a paradigm for a chronic rheumatic disease in which host immunogenetic susceptibility factors interact with a microbial trigger [1].
The dominant host factor is the class I HLA gene B27, which is present in 60-90% of such cases. The infections that initiate the disease are either Salmonella, Shigella, Campylobacter, and Yersinia spp., or infections with Chlamydia trachomatis. There have been numerous mechanisms postulated to explain this kind of arthritis, including molecular mimicry, immune response to arthritogenetic peptide, immune complex formation, in situ antigen deposition, and toxin-mediated synovitis [2].
All Chlamydia species are obligate intracellular bacterial parasites, and all are pathogenic to their various hosts [3]. A number of studies have also indicated that Chlamydophila (Chlamydia) pneumoniae is another, although less frequent, causative agent in arthritis [4,5].
Both species disseminate from their sites of primary infection, and when they do so, these organisms often take up long-term residence at distant anatomic locations. At sites of their dissemination, neither C. trachomatis nor C. pneumoniae produces any known toxins. Rather, both species may elicit a powerful immunopathogenic response that in turn can engender various diseases, one of which is inflammatory arthritis. Persistent chlamydial organisms exist in a morphologically aberrant, but metabolically active, state in synovial tissue [6,7].
Incidence of the disease is 5/100,000 patients aged 18-60 years [8]. A relatively small proportion of individuals who acquire a genital infection with C. trachomatis develop acute inflammatory (reactive) arthritis, and only a portion of those patients proceed to chronic disease [9].
Furthermore, serological tests can be helpful in establishing past or present chlamydial infections. Also, in chronically infected patients in whom the bacteria are no longer detectable locally in the synovium, a positive serological test may be the only indication of chlamydial involvement [14].

MATERIALS AND METHODS
This study was conducted on 33 known patients with chronic arthritis for > 6 months, with negative rheumatoid factor (RF) in their sera, aging from 26 to 61 years. Other 33 subjects, aging from 22 to 60 years with nearly similar proportion between genders, have enrolled in this study as a control group, selected blindly to their medical conditions (apart from chronic arthritis and similar conditions). Table 1 shows the distribution of cases according to age. More than half of the study group cases (about 57%) were in age between 30 years and 49 years old, with mean age of (46) years old. The mean age of the control group is (45) years old.
Among the 33 cases, 28 were females, and the other 5 were males (female: male ratio is 5.6:1). Fig. 1 illustrates the distribution of cases according to gender.
Base line data about subjects were obtained from their history and clinical examination, a previously arranged questionnaire was used for this purpose. Table 2 shows the main clinical manifestations of the chronic arthritis group in current study. Venous blood samples of about 5 ml were aspirated and collected in dry plain tubes. After taking part of blood to perform complete blood picture (CBP), erythrocyte sedimentation rate (ESR) and RF, blood in plain tubes allowed to clot (for minimum 30 minutes) at room temperature (20-25°C), then separated by centrifugation (3000 rpm for 5 minutes separate the serum and dispensed into sterile tightly closed Eppendrof tubes and stored at 20°C until assayed. All sera samples were tested for CBP, RF, and ESR. Septic arthritis was excluded from all cases by history, clinical examination, and lab investigations (CBP; with no signs of bacterial infection). Also, RA was excluded by history, clinical examination, along with the negative RF in all samples sera.

The distribution of study group cases according to gender
The criteria of inclusion and the clinical manifestations followed for selecting chronic arthritis cases in current study group [15] Criteria followed for selecting chronic arthritis cases General symptoms & signs of chronic arthritis metric mono, oligo or polyarthritis, often affecting the lower extremities, that is not resolved for at least Pain, Tenderness, Swelling, Morning stiffness, Limitation in movement, Deformity, and Fever. (inflammation of the site of insertion of ligaments, tendons, joint capsule or fascia to bone, that is not Venous blood samples of about 5 ml were aspirated and collected in dry plain tubes. After taking part of blood to perform complete blood picture (CBP), erythrocyte sedimentation rate (ESR) and RF, blood in plain tubes allowed to clot (for minimum 30 minutes) at room C), then separated by centrifugation (3000 rpm for 5 minutes) to separate the serum and dispensed into sterile tightly closed Eppendrof tubes and stored at -All sera samples were tested for CBP, RF, and ESR. Septic arthritis was excluded from all cases by history, clinical examination, and laboratory investigations (CBP; with no signs of bacterial infection). Also, RA was excluded by history, clinical examination, along with the negative RF The ESR values are illustrated in all cases show high ESR level (ESR than half of cases (52%) were having ESR value of less than 60.
Rheumatoid factor (RF) was tested using commercial Latex agglutination test method (Cortez Diagnostics Inc., California qualitative and semi-quantitative measurement (based on reaction between human IgG bound to biologically inert latex particles and rheumatoid factors in the test serum specimen).
Both IgM and IgG for Chlamydia trachomatis were tested in two separate ELISA kits (Novatec Diagnostica, Germany) using sandwich assay for the qualitative and quantitative determination of antibodies against C. trachomatis performed following the procedure protocol included within the kit packing as issued from the manufacturer company). The ESR values are illustrated in Fig. 2, where all cases show high ESR level (ESR ≥ 40). More than half of cases (52%) were having ESR value Rheumatoid factor (RF) was tested using commercial Latex agglutination test method (Cortez Diagnostics Inc., California-USA) for quantitative measurement sed on reaction between human IgG bound to biologically inert latex particles and rheumatoid factors in the test serum specimen).

Chlamydia trachomatis
were tested in two separate ELISA kits (Novatec , Germany) using sandwich assay for the qualitative and quantitative determination of (This test was performed following the procedure protocol included within the kit packing as issued from the

Fig. 2. The distribution of cases according to ESR values
Statistical analysis was done using GraphPad Software, (California, USA). Two samples unpaired T test was used to find the correlation between means of normally continuous samples of two groups of data. Findings with P value less than 0.05 were considered significant.

RESULTS AND DISCUSSION
The distribution of clinical manifestations among the study group is showed in Table 3. All the chronic arthritis patients were suffering from pain, and the majority (about 97%) were having morning stiffness.
Pain is a very clear complaint and it was annoying symptom to the vast majority of patients in this study, as it is shown in Table 3. Besides, the morning stiffness was also a well defined constitutional symptom in most of these cases. We suppose that it may be either a consequence of pain experienced during moving a joint, the symptom of loss of range of motion or the physical sign of reduced range of motion. Some articles and reports agreed with our finding in the present study [8,16]. The remaining clinical manifestations varied from fever to swelling and extra-synovial ones, that are reported in less percentages compared with the formal two.
The mean C. trachomatis IgM levels in sera of current arthritis cases are compared with those of the control group (Table 4). All cases of both groups were giving results below cut off value for the ELISA kit used. Non-significant relation regarding IgM levels was found between both groups in current study.
The mean C. trachomatis IgG levels in sera of current arthritis cases are also compared with those of the control group (Table 5). Although no cases were exceeding the cut off value of the ELISA kit used for IgG level in sera, but a significant relation (P<0.001) was found when using T-test to compare mean results in the two study groups.
Chlamydial infection causes serious sequelae, one of which is chronic arthritis. Reports indicate that approximately 5% of those with a Chlamydial infection will develop acute arthritis, and about half of these will proceed to chronicity [17]. Intraarticular persistence of viable, although nonculturable, C. trachomatis is considered to be the cause of arthritis [18].
In the current cross sectional study, the distribution of cases according to gender (Fig. 1) showed high female: Male ratio (5.6:1). As a probable explanation, this can be due to current cultural and social custom represented in delayed presentation of female cases and medical self-neglect in Iraqi population.
In Table 1, It is clear that more than half of cases (about 57%) were in age between 30 years and 49 years, with mean age of 46 years old. This clarifies the age range at which the patients suffering from chronic arthritis often present. They more often acquire the disease and seek medical consultation during this period. A number of studies [14,19,20] agreed with this finding.    As an inflammatory process, patients with chronic arthritis usually presented with elevated ESR; an obvious picture in this study (Fig. 2). In this work, we reported that 52% of patients were having ESR value of 40 -59 mm/hr. Yet, majority of the enrolled patients had high ESR.

No. of Patients
Furthermore, in order to maintain the inclusion criteria, we excluded septic arthritis from the studied sample by history, clinical examination, and laboratory investigations (CBP; with no signs of bacterial infection), as well as rheumatoid arthritis (RA) that was excluded by history, clinical examination, along with the negative RF in all samples sera. This exclusion helped in focusing on the disease in question; chronic aseptic arthritis, and facilitated achieving the aim of this study.
Specific serological tests were conducted in this study using ELISA. Both IgM and IgG for Chlamydia trachomatis were tested in patients' group and compared to those of the control subjects as well. As IgM levels are concerned (Table 4), the results showed a non significant difference between both groups in current study (P>0.05), which means that there is no considerable relation between the chronic arthritis and anti-Chlamydia trachomatis IgM levels.

CONCLUSION
This study showed that there is a significant statistical relationship between chronic arthritis and previous Chlamydia infection (demonstrated by high titer of anti-Chlamydia trachomatis IgG) among the patients enrolled in this work.

CONSENT
All authors declare that written informed consent was obtained from each patient before enrolment in this study for publication of this report.

ETHICAL APPROVAL
The study was approved by the Committee of Medical Microbiology Dept./ College of Medicine-Tikrit University for ethical consideration, and informed consent was obtained from all patients. This study is not against the public interest, or that the release of information is allowed by legislation.