Introduction 

At the end of the 20th century - the beginning of the 21st century, the results of several scientific studies conducted in Europe and North America proved that Chlamydia trachomatis is a trigger agent of reactive arthritis (ReA), seronegative spondyloarthritis (SpA) with a possible important role in undifferentiated seronegative oligoarthritis, undifferentiated monoarthritis syndrome and rheumatoid arthritis (RA) [2, 4, 9]. To date, clinical manifestations, changes in laboratory data and the results of instrumental examinations of joints and spine in axial seronegative spondyloarthritis (aSpA) with a trigger role of Chlamydia trachomatis have not been fully elucidated. The issues regarding antibacterial therapy and anti-inflammatory drugs in diseases of the joints and spine associated or induced by Chlamydia trachomatis are not still fully explored[1, 6, 7].

Purpose of the study: evaluation of clinical manifestations features, diagnosis, and evolution of seronegative spondyloarthritis associated with Chlamydia trachomatis infection.

Objectives of the study:

1.   To compare clinical manifestations, changes in laboratory parameters, results of instrumental examinations of the joints and spine in seronegative spondyloarthritis, the axial, peripheral, and mixed form. 

2.   To appreciate the significance of Chlamydia trachomatis in the development of SpA by detecting the presence of DNA using the polymerase chain reaction (PCR) in real time and by isothermal amplification of NASBA-PCR (Nucleic acid sequence-based amplification).

Material and methods

During the years 2015-2021, there were examined 138 patients with SpA at the „Timofei Mosneaga” Republican Clinical Hospital. Chlamydia trachomatis in the form of monoinfection was detected in 87 (63%) patients, of which 52 (59.8%) were women and 35 (40.2%) men. In 21 patients with SpA no concomitant infections were detected, while 30 patients had 2 and more concomitant infections.

The average duration of articular syndrome in all 108 patients before enrollment in the study was 2,0 (0,5-4,0) years. Depending on the diagnosis, the patients of the study group were divided into 4 subgroups. In order to identify the features of clinical manifestations, changes in laboratory parameters and the results of instrumental examinations of the joints and spine in patients with SpA associated with Chlamydia trachomatis infection, 4 comparison groups were created. The first group included 34 patients with peripheral SpA associated with Chlamydia trachomatis infection, the second group – 21 patients with axial SpA associated with Chlamydia trachomatis infection, the third group – 21 patients with SpA without concomitant infections and the 4th group – 32 patients with mixed form SpA associated with Chlamydia trachomatis infection.

The study groups were identical in terms of gender, age and duration of the disease. Clinical examination of patients included the collection of complaints, history, measurement of body temperature with an electronic thermometer, examination of organs and systems, evaluation of the severity of pain and disease activity using an analog visual scale (VAS) in mm, number of painful joints (NAD) and number of swollen joints (NAT) according to method 66/68, identification of joint deformities, as well as changes in periarticular tissues (entesopaties, bursitis, tendinitis and tenosynovitis).

The diagnosis was carried out using the New York and ASAS criteria [11, 13, 14]. Laboratory research methods included a complete blood count, biochemical tests with the determination of total proteins, glucose, total cholesterol, alanine aminotransferase, aspartate aminotransferase, uric acid, urea, creatinine, C-reactive protein (CRP), antistreptolysin-O and rheumatoid factor (RF), urinalysis, urine culture, HLA-B27 antigen. After examining the patients and obtaining the results of laboratory data, the disease activity index was calculated using the BASDAI index [12].

Diagnosis of Chlamydia trachomatis infection was carried out by the following methods: (1) DNA detection of the pathogen was carried out by scraping the epithelium from the urethra or cervical canal. (2) Determination of antibodies specific to IgM, IgA, IgG in the serum by enzymatic immunoassay (ELISA). Detection of diagnostic titers IgM and IgG, as well as IgA confirmed the presence of Chlamydia trachomatis infection. In addition patients with inflammatory diseases of the joints and spine, synovial fluid was tested with real-time PCR to identify fragments of 16S ribosomal RNA of the pathogen, indicating its persistence [3, 5, 8]. 

DNA from Trichomonas vaginalis, Ureaplasma urealyticum, Mycoplasma hominis was determined by PCR in real time.

Arthritogenic pathogens of intestinal infections (Yersinia, Shigella, Salmonella) were excluded by testing the patients’ serum for the presence of antibodies to these microorganisms[10]. Confirmation of the diagnosis, clarification of the stage of the disease and determination of the involvement of internal organs in the pathological process was carried out using instrumental methods: radiography of the affected joints and spine, computed tomography, magnetic resonance, ultrasound examination of the joints and internal organs, electrocardiography. For the processing of the obtained data, the STATISTICA 10.0 program was used. The normality of the distribution of quantitative traits was evaluated by the Kolmogorov-Smirnov method. The results of the normally distributed characteristics were presented as mean values (M) and standard error of the arithmetic mean (m). The comparison of the average values of such parameters in two independent groups was carried out using the unpaired Student t-test and in two dependent groups – the t-student pair test. Signs that did not comply with the law of normal distribution were shown as the middle and intertwines interval (Me, Q25-Q75).

In order to compare the parameters that did not comply with the law of normal distribution, the Mann-Whitney nonparametric test (U-test) was used in two independent groups. The comparison of the characteristics of the two dependent groups was carried out using the Wilcoxon test for pair comparisons (the W pair test). The qualitative characteristics have been described by absolute (n) and/or relative frequencies in percentages. Qualitative comparisons of independent groups were made using the Pearson χ2 test and the fisher exact test.

 The analysis of the relationship of qualitative characteristics was carried out by calculating the association coefficient Yule (Q). The differences obtained at all stages of the study were considered statistically significant at values of the materiality level reached p<0,05.

Results

Chlamydia trachomatis infection was detected in 78 (89.7%) of 87 patients examined by PCR, and in 9 (10.3%) cases by ELISA. Even though Chlamydia trachomatis is an intracellular pathogen, the DNA of the microorganism was found in the urethra/cervical canal in 7 patients, and in 5 of them the pathogen was identified in both urogenital scraping and synovia fluid.

Clinical, laboratory and instrumental characteristics of patients with the peripheral form of SA associated with Chlamydia trachomatis infection occur in most cases in young women (76.5%) and middle-aged women (91.1%).

The disease begins mainly with inflammation of the joints of the lower extremities (81.8%), of which the knee joints are mainly involved in the pathological process (63.6%). In less than half of patients (33.3%), arthritis occurs with subfebrility or fever. In the advanced stage of the disease (Me = 1,0 years), the clinical picture is dominated by oligo-polyarthritis (82,3%) with a predominant lesion of large joints (94,1%), of which the proportion of damage to the knee joints in the pathological process was the highest (73,5%). In patients with the peripheral form of SpA associated with chlamydial trachomatis infection, inflammation of the periarticular tissues (41.2%) with the appearance of talalgia, the development of Achilles’ bursitis, calcaneal spurs and tenosynovitis can also be observed.

An indirect sign of the association of arthritis with an infection is cutaneous hyperemia in the small joints of the hands and plants (23.5%). Extraarticular manifestations of the disease can also occur in the form of myalgia (8.8%). The peripheral form of SpA associated with Chlamydia trachomatis infection is not characterized by pronounced inflammatory changes in laboratory tests [Me = ESR 13,0 (7,0-28,0) mm/h, Me = CRP 3,3 (1,2-19,9) mg/L]. Early radiological signs of arthritis were detected only in 23.1% of the patients examined. The peripheral form of SpA associated with Chlamydia trachomatis infection differs from the peripheral form of SpA associated with Mycoplasma hominis, the latter having a significantly higher proportion of patients with polyarthritis at the onset of the disease, involvement in the pathological process mainly small joints of the upper extremities, namely, metacarpophalangeal joints and proximal interphalangeal joints of the hands. In the advanced stage of the disease, the differences in the clinical picture between both variants disappear. Laboratory and instrumental data of the disease are similar. Only in peripheral form of SpA associated with C. trachomatis there is a greater proportion of patients with an increase in C-reactive protein (U = 394,0; p = 0,036).

The axial form of SpA associated with Chlamydia trachomatis infection occurs mainly in young men (85.7%). In more than half of the patients, large joints are predominantly involved in the pathological process (58.8%), preferentially involving the lower extremities. The axial variant of the SpA can begin with an injury in any sector of the spine.

In the axial form of SpA form associated with Chlamydia trachomatis, the proportion of involvement in the pathological process of sacroiliac joints was 100%, large peripheral joints -71.4% and knee joints - 61.9 % being the highest. In axial form of SpA, fever can be detected, but in more than half of the patients it is absent (57.1%). Change in skin color on the joint area in the form of hyperemia or cyanosis (9.52%), as well as lesions of the eye membranes in the form of conjunctivitis, uveitis or iridocyclitis (6.3%) are observed in a small number of patients.

In axial form of SpA, the involvement of periarticular tissues in the pathological process in the form of enthesitis, perichondritis, Achilles and calcaneal spurs (38.1%) is present. Laboratory tests for axial SpA associated with Chlamydia trachomatis infection are characterized by an increase in the level of acute phase reactants of C-reactive protein 37,2 ± 7,02 mg/l and ESR 36,3 ± 7,57 mm/h, while white blood count in most cases does not exceed the upper limit (57,1%). For axial SpA associated with Chlamydia trachomatis infection, a marked activity of the inflammatory process is typical according to the results of the BASDAI index (5.9 ± 0.38).

According to instrumental methods of research, patients with axial SpA associated with Chlamydia trachomatis infection show both unilateral and bilateral lesions of the sacroiliac joints. In other sectors of the spine and peripheral skeleton, nonspecific radiographic manifestations of a chronic inflammatory process in the form of osteoporosis, wear, intraosseous cysts, as well as spondylitis, spondylodiscitis, perichondritis and syndesmophytosis can be found. At the onset of the disease, axial SpA associated with Chlamydia trachomatis infection does not differ in its clinical manifestations with SpA associated with other infections.

However, unlike SpA without concomitant infections in axial SpA associated with Chlamydia trachomatis, in a larger number of patients, small joints of hands and plants (F = 0.15; p = 0.022) are involved in the inflammatory process. In the advanced stage of the disease, axial SpA associated with Chlamydia trachomatis infection, in contrast to SpA without concomitant infections, is characterized by inflammation of the first metatarsophalangeal joint (F = 0.13; p = 0.023), predominantly unilateral lesion of the sacroiliac joints (F = 0.15; p = 0.021), recurrent febrile syndrome (F = 0.1; p = 0.042) and higher laboratory activity of the disease in terms of ESR (p = 0.043) and C-reactive protein (p = 0.031). 

Discussions 

Using modern methods of molecular genetic diagnostics (real-time PCR to identify the DNA of the pathogen), it has been established that Chlamydia trachomatis can be an infectious trigger of SpA. The latter guides the clinical manifestations and the course of this disease. This research describes the previously unidentified clinical, laboratory and instrumental features of SpA associated with Chlamydia trachomatis infection. 

In patients with SpA, Chlamydia trachomatis infection is usually found only in the urogenital tract. The presence of Chlamydia trachomatis infection in patients with ankylosing spondyloarthritis influences the clinical manifestations of the disease. The peripheral form of SpA associated with Chlamydia trachomatis infection is characterized by the onset of the disease after a previous urogenital infection. Damage to the elbow joints, metacarpophalangeal and metatarsophalangeal joints is not typical. Axial spondyloarthritis associated with Chlamydia trachomatis infection, in contrast to spondyloarthritis due to infections, is characterized by the involvement of especially small joints of hands and plants in the inflammatory process at the onset of the disease. Damage to the first metatarsophalangeal joints is also common, and fundamentally is the unilateral inflammation of the sacroiliac joints, recurrence of febrile syndrome, and a higher activity of the disease expressed by laboratory data. The results of the research data contain new and relevant information to improve the quality of diagnosis of patients with SpA associated with Chlamydia trachomatis infection.

Conclusions

1.      Chlamydia trachomatis infection was detected by real-time polymerase chain reaction, and antibodies to it in the diagnostic titer – by immuno-enzymatic test for patients with SpA, in patients with and without concomitant arthritogenic infections. With the help of PCR reaction, this pathogen was determined in the urogenital tract (89,7%) and by ELISA (10,3%). In the synovial fluid of patients with SpA peripheral form, not only the DNA of the pathogen, but also the ribosomal RNA of Chlamydia trachomatis was detected by isothermal amplification of nucleic acids, which indicates the viability and low metabolic activity of the pathogen in the affected joint cavity.

2.      SpA associated with Chlamydia trachomatis infection has more expressed clinical, laboratory and instrumental manifestations both at the onset and in the advanced stage of the disease. 

3.      Peripheral form of SpA associated with Chlamydia trachomatis infection is characterized by the appearance of polyarticular syndrome from the onset of the disease, with mainly damage to the small joints of the extremities, among which metacarpophalangeal joints and proximal interphalangeal joints of the hands are most often involved in the pathological process. For peripheral form of SpA associated with Chlamydia trachomatis infection, the presence of pronounced inflammatory changes in serum is not typical [the protein C-reactive level 3.3 (1.2-19.9) mg/l].

4.      SpA axial form associated with Chlamydia trachomatis infection, in contrast to spondyloarthritis without concomitant infections, is characterized by the involvement of small joints of the hands and plants in the pathological process at the onset of the disease. With the development of spondyloarthritis associated with Chlamydia trachomatis infection, in comparison with spondyloarthritis without concomitant infections, a significantly higher number of patients had involved in the pathological process the first metatarsophalangeal joint, unilateral lesions of the sacroiliac joints, recurrence of febrile syndrome, greater laboratory activity of the disease: the level of ESR and the level of CRP being increased.

5.      SpA mixed form associated with Chlamydia trachomatis predominantly is characterized by the onset of the disease after a previous urogenital infection. In the advanced stage of the disease, damage to the elbow joints, metacarpophalangeal and metatarsophalangeal joints is not typical.

Authors contribution 

Conception and design: LC, LG. Accumulation of raw data: LC, LG, ER, VC. Analysis and interpretation of data: LC, ER. Manuscript development: LC. Manuscript review: LC, LG. All authors have read and approved the final version of the manuscript.

Authors’ ORCID IDs

Lia Chișlari - https://orcid.org/0000-0002-7088-568X

Liliana Groppa - https://orcid.org/0000-0002-3097-6181

Eugeniu Russu - https://orcid.org/0000-0001-8957-8471

Victor Cazac- https://orcid.org/0000-0001-9293-4481

Larisa Rotaru - https://orcid.org/0000-0002-3260-3426

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