Temporomandibular joint (TMJ) involvement in juvenile idiopathic arthritis (JIA) occurs in up to 80% of affected children. The purpose of this study was to investigate the presence of bacterial DNA in synovial fluid, and to compare this with clinical and immunological findings in children with JIA, adults with persistent JIA, and adults with rheumatoid arthritis, in order to detect whether bacteria contribute to inflammation in TMJ arthritis. Synovial fluid and skin swab samples were collected from 30 patients (54 TMJs). Bacterial detection was performed using 16S rRNA pyrosequencing. Bacterial DNA was detected in 31 TMJs (57%) in 19 patients (63%). A positive statistically significant correlation was registered between bacterial DNA detected in TMJ synovial fluid and the following factors: total protein concentration in synovial fluid, interleukin 1β, tumour necrosis factor alpha, adrenocorticotropic hormone, and adiponectin, as well as the duration of the general medical disease. Fourteen different bacterial species were detected in synovial fluid. Bacterial DNA in TMJ synovial fluid without contamination was detected in more than 50% of the patients. Studies are needed to evaluate the consequences of this bacterial DNA in synovial fluid with regard to TMJ arthritis.
Juvenile idiopathic arthritis (JIA) is a broad term that describes a clinically heterogeneous group of inflammatory joint diseases of unknown cause, with an onset before 16 years of age. It was first described by Still in 1897. Temporomandibular joint (TMJ) involvement occurs in up to 80% of children with JIA, but is asymptomatic in 70% of these children.
Rheumatoid arthritis (RA) is a chronic, inflammatory condition that mainly affects joints. It is associated with pain, erosion, disability, and reduced survival. TMJ symptoms may occur in 65% of patients with RA.
Synovial fluid contains hyaluronic acid and interstitial fluid filtered from blood plasma, and it was previously believed to be aseptic. Studies on the large joints, excluding the TMJ, have detected Salmonella spp., Shigella spp., Mycobacterium tuberculosis , and Campylobacter spp. in patients with juvenile onset spondylo-arthro-pathologies. Other studies have detected Borrelia burgdorferi and Yersinia spp. With regard to the TMJ, studies have detected bacteria in synovial fluid in both healthy TMJs and TMJs with disc displacement, and the non-healthy joints have shown a higher prevalence of bacteria and of Staphylococcus aureus in particular. Other researchers have detected bacteria only in TMJ synovial fluid from patients with a temporomandibular disorder (TMD), while the healthy joints have been found to be aseptic. These studies have reported a wide variety of bacterial species in the synovial fluid of the TMJ. However, little is known about the presence of bacteria in TMJ synovial fluid in TMJ arthritis.
The aetiology of TMJ arthritis in JIA and RA is not fully understood and several studies have been performed to locate a factor that triggers inflammation.
The purpose of this study was to investigate the presence of bacterial DNA in synovial fluid and to compare this with clinical and immunological findings in children with JIA, adults with a persistent JIA, and adults with RA, in order to ascertain whether bacteria are contributors to inflammation in TMJ arthritis.
Materials and methods
Synovial fluid was taken from the TMJs of 30 patients, 24 bilaterally and six unilaterally for a total of 54 joints. Twenty patients (35 TMJs) were children with JIA, five patients (nine TMJs) were adults with previous JIA (AJIA) continuing into adulthood, and five patients (10 TMJs) had RA. Clinical findings in the JIA patient cohort have been published previously.
Clinical examinations were performed before synovial fluid collection and at two follow-up appointments (3 and 8 months). The results of these examinations have been described previously. They included recordings of maximum incisal opening (MIO), pain incisal opening (PIO), lateral excursion, and pain upon palpation of muscles and the TMJs, as well as subjectively reported pain and function of the jaw (visual analogue scale, VAS). Bacteria were detected in synovial fluid in 19 patients. Six patients (12 TMJs; three JIA, one AJIA, and two RA) were excluded from the study due to possible contamination.
This prospective clinical trial of arthrocentesis of the TMJ in patients with JIA, AJIA, and RA was approved by the regional medical ethics committee.
Samples were collected from the TMJ using a push and pull technique, as previously used by Alstergren et al. and in a recently published study. Ultrasound-guided sampling was performed in an operation field after prior disinfection of the skin in an area of 3 cm surrounding the penetration site; this was done using five washes of chlorhexidine–ethanol 5 mg/ml solution (Klorhexidinsprit 5 mg/ml; Fresenius Kabi, Nydalen, Norway) in accordance with the recommendations of Oslo University Hospital. Skin swabs were taken from both the disinfected skin and non-disinfected skin in the puncture area prior to sampling, using a sterile Whatman OmniSwab (GE Healthcare, USA). All samples were frozen immediately at −80 °C.
DNA isolation, PCR amplification, and sequencing
The extraction of genomic DNA was performed using a MasterPure Complete DNA Purification Kit (Epicentre Biotechnologies, Madison, WI, USA), based on the manufacturer’s extraction protocol for fluid samples.
PCR amplification was performed using 16S rRNA gene primers flanking the V3 and V5 region: E334F 5′-CCAGACTCCTACGGGAGGCAGC-3′ and E939R 5′-CTTGTGCGGGCCCCCGTCAATTC-3′. The PCR amplification steps included a denaturation step at 96 °C for 2 min and 32 amplification cycles: 96 °C for 30 s, 61 °C for 40 s, 72 °C for 30 s.
Amplicons were gel-purified (GenElute, NA1111; Sigma Aldrich) and re-sequenced with fusion primers, Roche 454 FLX adaptor primers, 8-base bar code in front of E334F, reverse adaptor primer (B) fusion with E939, and AMPure bead purification (Agencourt AMPureXP beads, A63880; Beckman Coulter, Inc.) of fusion PCR products, and were quality check by Agilent Bioanalyzer high sensitivity chip (Agilent High Sensitivity DNA, catalogue No. 5067-4627; Agilent Technologies, Inc.).
Amplicons were sequenced using a GS-FLX 454 sequencer (454 Life Sciences, Roche) at the Norwegian Sequencing Centre, Centre for Ecological and Evolutionary Synthesis, University of Oslo, Norway. Sequences (reads) were edited using the Ribosomal Database Project (RDP) pipeline and mothur software. Chimera UCHIME and reference 16S rRNA ( www.arb-silva.de ) were used before trimmed FASTA sequences were blasted using the Bioportal at the University of Oslo. MEGAN (Centre for Bioinformatics ZBIT, Tübingen University, Germany) was used to visualize the clustering and taxonomy of the samples.
Statistical analyses of the correlation between clinical data, immunological data, and bacteria detection were performed using Sigma Plot 12.0 for Windows (Systat Software, Inc., San Jose, CA, USA). Parametric and non-parametric statistics ( t -test and Wilcoxon test) were performed for patients with either one or two TMJ synovial fluid samples positive for bacteria detection, and to compare patients with and without the presence of bacteria in the TMJ synovial fluid.
Bacterial DNA was detected in nine patients with JIA (12 TMJs), three patients with AJIA (five TMJs), and in one patient with RA (two TMJs) – in all 19 TMJs in 13 patients. Clinical data for these patients are shown in Table 1 . There was no statistically significant difference between patients with one or two TMJs with a positive PCR product.
|Joint a||JIA||RA||Age (years)||MIO (mm)||Lateral excursion b||Pain VAS c||Function VAS c||JIA/RA disease duration (years)||Duration of symptoms from the TMJ (years)|
The detection of bacterial DNA (positive 16S rRNA PCR product) in TMJ synovial fluid correlated significantly with the total protein concentration in synovial fluid ( P < 0.05), interleukin 1β (IL-1β; P < 0.005), tumour necrosis factor alpha (TNF-α; P < 0.01), adrenocorticotropic hormone (ACTH; P < 0.005), and adiponectin ( P < 0.05). There was a positive correlation between synovial fluid bacteria detection and the duration of the general medical disease ( P < 0.05), while there was a negative correlation between synovial fluid bacteria detection and age ( P < 0.05), increased subjective pain (VAS, P < 0.05), jaw function (VAS, P < 0.05), and with pain on palpation of the TMJ (VAS, P < 0.05).
On examination of each bacterial species detected, there was a positive correlation between Propionibacterium and IL-6 and pain on palpation of the TMJ ( P < 0.05), and between Streptococcus and IL-1 ( P < 0.05). Each bacterial species detected in the TMJ synovial fluid is presented in Table 2 as a proportion (%) of the total bacteria detected. The most frequent bacterial species was Pseudomonas fluorescens . In all, 14 different bacterial species were detected in the synovial fluid.
|Joint a||Propionibacterium spp.||Pseudomonas spp.||Pseudomonas F||Porphyromonas spp.||Streptococcus spp.||Acidobacteria spp.||Veillonella spp.||Rhizobial spp.||Corynebacterium spp.||Actinomyces spp.||Leptothrixaea spp.||Jonquetella spp.||Tannerella spp.||Clostridium spp.||Unclassified|