International Journal of Rheumatic Diseases 2014


Chlamydia trachomatis elementary bodies in synovial fluid of patients with reactive arthritis and undifferentiated spondyloarthropathy in India Praveen KUMAR,1 Geetika KHANNA,2,3 Sumit BATRA,2 Vinod K. SHARMA2,3 and Sangita RASTOGI1 1

Microbiology Laboratory, National Institute of Pathology (ICMR), 2Central Institute of Orthopedics (CIO), and 3Vardhaman Mahavir Medical College (VMMC) and Safdarjung Hospital, New Delhi, India

Abstract Objectives: Reportedly, there is little information on the magnitude of genitourinary-induced reactive arthritis (gReA) from India. Genital infection with Chlamydia trachomatis is a major health problem in India because of its high prevalence; therefore, this study was conducted with the aim to screen ReA/undifferentiated spondyloarthropathy (uSpA) patients (n = 20) attending a major city hospital in New Delhi, for investigating the presence of intra-articular chlamydial antigen in knee joints. Patients with rheumatoid arthritis (RA) and osteoarthritis (OA) served as controls (n = 20). Methods: Synovial fluid samples were screened for chlamydial elementary bodies (EBs) using a commercial kit (MicroTrak C. trachomatis Direct Specimen Test; Trinity Biotech, USA) for performing direct fluorescence assay (DFA). Results: Chlamydia trachomatis EBs were detected in the synovial fluid cell deposits of six patients in Group I, namely, 33.3% (4/12) ReA and 25% (2/8) uSpA. All C. trachomatis positive patients exhibited an oligoarticular clinical picture with knee joint involvement. In the synovial fluid cell deposits of control patients, namely, RA/ OA, no chlamydial EBs could be detected. Conclusions: This is the first study reporting the presence of C. trachomatis EBs in the synovial fluid of spondyloarthropathy patients, namely, ReA/uSpA from our country and it can be concluded that the prevalence of C. trachomatis-induced ReA is underestimated. Although our study had limitations in terms of sample size and lower sensitivity of DFA, yet this test can be used as an initial diagnostic tool for screening and patients with positive results may undergo specific tests for validation. Key words: Chlamydia trachomatis, elementary body, reactive arthritis, synovial fluid, undifferentiated spondyloarthropathy.


Correspondence: Dr Sangita Rastogi, MSc, MPhil, PhD, Microbiology Laboratory, National Institute of Pathology (ICMR), Safdarjung Hospital campus, Post Box No. 4909, New Delhi110 029, India. Email: [email protected]

The obligate intracellular pathogen Chlamydia trachomatis has emerged as a major causative agent in genitourinary reactive arthritis (gReA) in various countries.1,2 Furthermore, it was reported that C. trachomatis is present in metabolically active form during the remitting phase in synovial tissues from patients with chronic Chlamydia-induced reactive arthritis.3 Also, genitourinary

© 2014 Asia Pacific League of Associations for Rheumatology and Wiley Publishing Asia Pty Ltd

P. Kumar et al.

infection with C. trachomatis precedes sexually acquired ReA in approximately 50% of cases.4 Patients testing negative for genitourinary infection with C. trachomatis have also been detected with chlamydial infection in their joints. Approximately 30% of ReA patients do not have a preceding symptomatic infection.5 In developing countries, enterically acquired ReA is more common and studies from India have also largely focused on patients with ReA due to enteric pathogens6,7 and there is a paucity of data on gReA. In an earlier study reported from India, the clinical profile and prevalence of serum immunoglobulin A (IgA) antibodies against a panel of bacterial antigens, including C. trachomatis, in the sera of patients with ReA was reported.8 Another study from north India reported the prevalence of serum IgA antibodies to C. trachomatis in gReA patients as 25%.9 As a high prevalence of genitourinary infection with C. trachomatis has been reported in India,10–19 there is a possibility that the association of C. trachomatis in gReA may well be currently overlooked and there is a need to uncover the magnitude of chlamydial infection in such patients. Chlamydiae presumably reach the joint via extravasated infected monocytic cells originating at the site of initial infection. Various molecular as well as nonmolecular techniques have been used for diagnosis of C. trachomatis in the synovial joints of patients with ReA.20,21 Although the isolation of C. trachomatis by culture has been largely unsuccessful in ReA,22 C. trachomatis DNA/RNA/antigen/proteins have been detected in the joints.23–25 Polymerase chain reaction (PCR) is considered to be the most promising tool for routine diagnosis of C. trachomatis infection, yet the results vary between different laboratories. Further, the method used for detection of C. trachomatis in the synovium is still a matter of choice as various workers have used different methodologies for detection in various settings and there is no uniformity with regard to sampling and methodology used for detection. In this regard, the use of direct immunofluorescence assay for detection of C. trachomatis probably stands out as a more convenient and cost-effective method of initial diagnostic testing and may assist in the detection of C. trachomatis in instances where specimens have lost viable chlamydiae through prolonged transport or suboptimal storage.26 It may be particularly useful during preliminary investigation of patients with a clinical picture of ReA as it is less time-consuming method. Hence, our study focused on screening gReA and undifferentiated spondyloarthropathy (uSpA) patients attending a


major city hospital in New Delhi (India), for the presence of C. trachomatis antigen in the synovial fluid.

MATERIALS AND METHODS Study subjects After taking informed written consent from each patient, a total of 50 age-matched arthritic patients, namely spondyloarthropathy (ReA/uSpA), rheumatoid arthritis (RA) and osteoarthritis (OA) patients presenting at Central Institute of Orthopedics (CIO), Safdarjung Hospital, New Delhi, India were enrolled in consultation with the clinician. However, only 20 patients with ReA/uSpA, 13 with RA and seven with OA were finally included in the study as the remaining 10 patients did not provide consent for arthroscopy, which is an invasive procedure for aspiration of the joint fluid. ESSG (European Spondyloarthropathy Study Group) and ACR (American College of Rheumatology) criteria were followed for selection of ReA/uSpA and RA patients, respectively.27,28 Both male and female patients were included. The study had the permission of Safdarjung Hospital Ethics Committee. At the time of enrolment, detailed clinical history was recorded for each patient, including history of tuberculosis or any other extra-articular feature. Patients with ankylosing spondylitis, inflammatory bowel disease, psoriasis and with history of preceding enteric infection, were excluded. Also, patients who received antibiotic treatment in the previous 3 months were not included in the study. Patients were divided into two groups, namely Group I (n = 20) comprised of ReA and uSpA patients having pain in single or multiple joints with/without signs and symptoms of urogenital infection. Among these, 12 were patients with ReA while the remaining eight were uSpA patients. Patients with RA and OA constituted the inflammatory and non-inflammatory controls, respectively in Group II (n = 20).

Collection of clinical samples Synovial fluid (2–5 mL) was aspirated under local anesthesia from the knee joint during arthroscopy from each patient (Groups I and II) and immediately transferred in sterile vials for transportation to the laboratory. Also, male/female genitourinary swabs from each patient were collected in phosphate-buffered saline (PBS).

Immunofluorescence microscopy After centrifugation at 600 9 g for 10 min, the cell deposit from the genitourinary swab/synovial fluid was

International Journal of Rheumatic Diseases 2014

C. trachomatis in reactive arthritis

spun onto clean glass microscope slides using Cytospin (Shandon, Astmoor Run, UK). Four slide preparations were made from each sample for detection of C. trachomatis antigen by direct fluorescence assay (DFA) using MicroTrak C. trachomatis Direct Specimen Test (Trinity Biotech, Jamestown, NY, USA) as per the manufacturer’s recommendations. Briefly, slides were fixed in methanol and subsequently incubated at room temperature with fluorescein isothiocyanate-conjugated monoclonal antibody to C. trachomatis major outer membrane protein (MOMP). Evans blue dye was used as control for autofluorescence while fluorescein isothiocyanate-conjugated monoclonal antibody to herpes simplex was used as another control antibody. For confirmation of morphology, a total of four slides were prepared for each patient. Each slide was read by two independent observers after blinding. Each observer counted elementary bodies (EBs) in five fields per slide using 100 9 oil objective and calculated the average number of EBs. Diagnosis of C. trachomatis was based on the presence of a minimum of seven or more chlamydial EBs per slide which appeared as pinpoints of bright green fluorescence. Likewise, enumeration and an average count of EBs were made by each observer in each of the four slides.

Statistical analysis Statistical analysis was performed with GraphPad Prism software (version 5.0; GraphPad Software, Inc., San Diego, CA, USA). Fisher exact test was performed for different variables. Mean and standard deviation were derived for numerical data using Column statistics. The inter-observer findings of each observer were analyzed and the degree of agreement was assessed by performing Kappa statistics.29 The Kappa values ranging from 0.01

to 0.20 were considered as slight agreement, from 0.21 to 0.40 as fair agreement, from 0.41 to 0.60 as moderate agreement, from 0.61 to 0.80 as substantial agreement and from 0.81 to 0.99 as almost perfect agreement.

RESULTS The range of EBs seen in the synovial fluid cell deposits by each observer varied from at least a minimum of seven and maximum of 10–15 EBs per slide. Among ReA/uSpA patients (n = 20), six (30%) were found to be C. trachomatis-positive, 12 (60%) were negative, while two (10%) patients could not be categorized as positive/negative. Hence, Kappa statistics were applied for inter-reader agreement and an overall good agreement (j = 0.780; 95%CI: 0.651–0.909) was seen between both observers. Among six infected patients with ReA/uSpA in Group I, 33.3% (4/12) had ReA while 25% (2/8) were uSpA patients. Of these, five (three ReA; two uSpA) male patients (83.3%) and one female ReA patient (16.6%) were C. trachomatis-positive. All C. trachomatis-positive patients (n = 6) exhibited an oligoarticular clinical picture with knee joint involvement. The clinical and demographic profile of arthritic patients has been summarized in Table 1. Among six male/female patients detected with chlamydial antigen in joints, three male ReA patients had urethritis while one female ReA patient was diagnosed with cervicitis. Both uSpA patients had no urogenital manifestations (Table 2). During genitourinary swab examination by DFA, two ReA patients (one male and one female) were found positive for chlamydial EBs. Both these patients were also positive for chlamydial EBs in the synovial fluid (Table 2).

Table 1 Clinical and demographic data of arthritic patients Characteristics M : F ratio (%) Mean age  SD Mean disease duration  SD (months) Monoarthritis (%) Oligoarthritis (%) Polyarthritis (%) Mean CRP (lg/mL) RF factor (%)





(n = 12) 9 : 3 (75 : 25) 31.1  6.8 15  9.9

(n = 8) 6 : 2 (75 : 25) 30  9.2 14.14  9.8

(n = 13) 2 : 11 (15 : 85) 36.6  7.7 36  16.4

(n = 7) 2 : 5 (29 : 71) 36  6.7 40  14.4

1 (8.3) 8 (66.6) 3 (25) 39.9  36.8 0 (0)

0 (0) 6 (75) 2 (25) 28.9  17.1 0 (0)

0 (0) 1 (7.6) 12 (92.3) 27.7  16.3 13 (100)

1 (14.2) 6 (85.7) 0 (0) 6.3  0.1 0 (0)

ReA, reactive arthritis; uSpA, undifferentiated spondyloarthropathy; RA, rheumatoid arthritis; OA, osteoarthritis; CRP, C-reactive protein; RF, rheumatoid factor.

International Journal of Rheumatic Diseases 2014



+ – – + – – Urethritis Urethritis Urethritis Cervicitis Nil Nil + – + + + – – + – – + + Knees, Ankles Knees, Ankles Knees, Shoulders, Hips Knees Knees, Ankles Knees O O O O O O 40/M 32/M 38/M 32/F 23/M 21/M 1/ReA 2/ReA 3/ReA 4/ReA 5/uSpA 6/uSpA

ReA, reactive arthritis; uSpA, undifferentiated spondyloarthropathy; M, male; F, female; O, oligoarticular. †Diagnosis of C. trachomatis by direct fluorescence assay using MicroTrak C. trachomatis Direct Specimen test.

– – – – – –

Buttock pain Sacroiliitis/ conjunctivitis Low back ache Morning stiffness Joints involved Joint pattern Age (years)/sex Patient no./diagnosis

Table 2 Clinical profile of Chlamydia trachomatis-positive spondyloarthropathy patients (n = 6)

ReA occurs worldwide, affecting young adults in the 20–40 years age group, with a male-to-female ratio of 3 : 1.7 Approximately 4–15% of those with genital C. trachomatis infections subsequently develop arthritis.30 The diagnosis of Chlamydia-associated ReA was initially based on the culture of the microorganism from urogenital samples. However, the demonstration of C. trachomatis in the urogenital tract alone cannot prove the chlamydial etiology of arthritis and intraarticular chlamydial detection is generally performed.31 In the present study, 2/6 patients who were positive in synovial fluid were also positive on genitourinary swab; however, it is uncertain whether these patients had C. trachomatis infection in the past as antibody detection was not performed. Also, 4/6 patients were found to be negative in genitourinary samples but positive in synovial samples. This is not unusual as the primary infection of the urogenital system is often cleared by the immune system or antibiotic treatment or both. Second, the initial inflammatory response in the genital system elicited by the infection attracts mononuclear cells which become infected, are subsequently extravasated, and via the general circulation make their way to the joint. Direct fluorescence cytology has enabled chlamydiae to be detected in the joints of patients with sexually acquired ReA using MicroTrak C. trachomatis Direct Specimen Test kit. This kit is used for the detection of C. trachomatis in urogenital/rectal/conjunctival/nasopharyngeal swab specimens but several groups of investigators have used this commercial kit to identify chlamydial EBs/inclusion bodies in synovial fluid/ biopsy specimens from patients with ReA.32–34 Interestingly, in a study published in Lancet, 80% concordance was reported between direct immunofluorescence and PCR results.23 These authors suggested that the discordant results in the remaining 20% of patients could be due to loss of chlamydial EBs during storage or in the process of transfer from slide to PCR reaction mixture. Recent studies have demonstrated that biopsy material from the synovial membrane of patients with Chlamydia-induced arthritis often contains intact chlamydial nucleic acids, even as late as 12 years after the onset of

Urogenital manifestations


– – – – – –

Urogenital C. trachomatis†

C. trachomatis† in joints

In the synovial fluid cell deposits of controls, namely RA/OA patients (Group II), no chlamydial EBs could be detected. None of the ReA/uSpA/OA patients were positive for rheumatoid factor while all RA patients were found to be positive (Table 1).

+ + + + + +

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International Journal of Rheumatic Diseases 2014

C. trachomatis in reactive arthritis

disease.24,35 Furthermore, morphologically atypical, but intact-appearing chlamydial organisms were found in the phagosomes of monocytes and to a lesser extent in synovial fibroblasts, suggesting that C. trachomatis can persist in the joint in a viable state.35 Once Chlamydia enters the persistent state, it cycles between an aberrant arrested state and replication, and the typical acute lifecycle is downregulated. A major issue relating to persistent chlamydial infection of the joint concerns the means by which the pathogen reaches the synovium from the urogenital system, the usual site of primary infection. Intracellular Chlamydia has been demonstrated within monocytes isolated from synovial fluid and from the synovial membrane of patients with Chlamydia-induced arthritis. These observations suggest that monocytes are involved as the vehicle transporting chlamydiae from the genital epithelium to the synovium. Theoretically, it can be speculated that chlamydiae persist mainly in the urogenital tract and that the demonstration of viable chlamydiae in the joint reflects continuous/discontinuous dissemination via circulation3,36 than persisting infection of the joint.37 In persistent stage, C. trachomatis might also display an aberrant morphology in the joints of chronic/acute patients with reactive arthritis. This is also attributed to the problem that direct detection at the primary infectious site is often not possible due to the late presentation of the patient and/or due to the fact that other tests such as serology have a low sensitivity.38 One of the limitations of this study is that some C. trachomatis-negative fluid samples might have been C. trachomatis-positive if synovial tissue were analyzed. Further, the DFA method by itself does not distinguish between infection-related disease versus a more classic molecular mimicry. Our findings are in agreement with other researchers who also detected the presence of chlamydial EBs in the joints by using immunofluorescence technique.32,34,39 It can be concluded that immunofluorescence cytology can be used for an intra-articular diagnosis of C. trachomatis during initial screening of ReA/uSpA patients. However, confirmatory diagnosis by nucleic acid amplification techniques such as PCR should be done in patients found to be C. trachomatis-positive by DFA.

ACKNOWLEDGEMENTS PK gratefully acknowledges the award of Senior Research Fellowship (SRF) by Indian Council of Medical Research, New Delhi (India).

International Journal of Rheumatic Diseases 2014

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International Journal of Rheumatic Diseases 2014

Chlamydia trachomatis elementary bodies in synovial fluid of patients with reactive arthritis and undifferentiated spondyloarthropathy in India.

Reportedly, there is little information on the magnitude of genitourinary-induced reactive arthritis (gReA) from India. Genital infection with Chlamyd...
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