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Rheumatology 2001; 40: 1355-1358
© 2001 British Society for Rheumatology

Original Papers

The prevalence of Mycoplasma fermentans in patients with inflammatory arthritides

C. B. Gilroy*, A. Keat1, and D. Taylor-Robinson

Department of Genitourinary Medicine, Medicine A, Imperial College School of Medicine, St Mary's Campus, Paddington, London W2 1NY and
1 Department of Rheumatology, Northwick Park & St Mark's Hospitals, Watford Road, Harrow, Middlesex HA1 3UJ, UK


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives. To search for evidence that Mycoplasma fermentans is involved in the pathogenesis of some forms of human arthritis by testing for the presence of mycoplasmal DNA in joint material.

Methods. M. fermentans DNA was detected by the identification of a 104-base pair amplification product of the polymerase chain reaction (PCR).

Results. M. fermentans DNA was detected in synovial fluid samples from six (17%) of 35 patients with rheumatoid arthritis (RA) and 18 (21%) of 85 patients with seronegative arthritis. These detection rates were significantly greater than in samples from patients with osteoarthritis or crystal synovitis, none of 26 of these being positive.

Conclusions. M. fermentans could be involved in the pathogenesis of some forms of inflammatory arthritis and this possibility is worthy of further study.

KEY WORDS: Mycoplasma fermentans, Inflammatory arthritis, Rheumatoid arthritis.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Mycoplasmas have been incriminated as causes of arthritis in many animal species. In some, the condition induced is acute and purulent, as in the case of Mycoplasma arthritidis-induced arthritis in rats [1]. However, in other species a chronic or relapsing polyarthritis develops. The chronic, severe and destructive polyarthritides due to M. hyosynoviae [2] or M. hyorhinis [3] in pigs and to M. capricolum in goats and sheep [4, 5] have often been compared with human rheumatoid arthritis (RA) [6]. Whether or not such comparisons are appropriate, there is no doubt that the large body of evidence for mycoplasmas causing arthritis in animals suggests that they should at least be considered as candidates for causing human disease. In this regard, mycoplasmas, including Ureaplasma urealyticum (ureaplasmas), have been associated with Reiter's disease [7, 8] and transient arthritic symptoms have been recorded during M. pneumoniae infections [9]. Furthermore, there is undisputed and convincing evidence for the involvement of mycoplasmas in arthritis in immunocompromised individuals [10, 11]. Ureaplasmas were first isolated from the joints of hypogammaglobulinaemic patients who had septic arthritis [10]. In addition, M. salivarium was isolated several times from the arthritic knee joint of a hypogammaglobulinaemic patient [12]. Furthermore, M. pneumoniae and M. hominis have been isolated from the joints of immunocompromised patients [13–15]. It is reasonable to conclude that these mycoplasmas have disseminated from the primary sites of infection, namely the respiratory tract for M. pneumoniae and the genital tract for M. hominis and ureaplasmas, the deficient immune system being unable to curtail the spread of the microorganisms. In such opportunistic infections, failure to isolate other agents and a rapid clinical response to anti-mycoplasmal antibiotic therapy provide support for the conclusion that mycoplasmas isolated from the joints are responsible for the arthritis [6].

In contrast, the role of mycoplasmas in RA is controversial. Early work suggesting a link between mycoplasmal infection and human RA was unconvincing [1, 6, 16]. Williams implicated M. fermentans as a cause of RA on the basis of its purported isolation from the synovial fluid of a large proportion of patients with this condition [17] and the inhibition of migration of leucocytes from patients with RA by membrane fragments of M. fermentans [18]. However, although M. fermentans has been isolated subsequently from the joints of a very small proportion of patients with RA [19–21], numerous attempts to repeat the original observation failed [22]. In addition, M. fermentans seemed an unlikely candidate because, at the time, it was isolated only rarely from the genitourinary tract [23].

The advent of molecular techniques, particularly the use of the polymerase chain reaction (PCR) is, however, providing new insights. Thus, by the use of the PCR, M. fermentans has been found in the throats of more than 20%, the peripheral blood leucocytes of about 10% and the urine of 5% of both HIV-positive and HIV-negative patients attending a genitourinary medicine clinic [24]. This was a compelling reason to re-evaluate the possible presence of M. fermentans in the joints of patients with RA using the PCR. Some data are already available, though they are conflicting. Schaeverbeke et al. [25] detected this mycoplasma in the joints of about 20% of French patients with chronic inflammatory arthritides, including RA, but not in those of patients with other joint diseases. On the other hand, Hoffman et al. [26] in the USA failed to detect M. fermentans in the joints of any patients with rheumatoid disease. Clearly, observations on more patients, with a range of diagnoses, in different geographical locations and with tests in different laboratories are warranted. In view of this, we examined specimens from the joints of British patients with chronic inflammatory arthritides and also from patients with osteoarthritis (OA) and acute crystal arthritis.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Synovial fluid (SF) samples from 146 patients in six diagnostic categories were examined (Table 1Go). A single SF sample from a knee joint of each patient was tested. Patients were categorized as having RA, undifferentiated seronegative arthritis (negative test for rheumatoid factor), psoriatic arthritis, reactive arthritis, OA or crystal synovitis (mainly gout) and their demographic details are summarized in Table 1Go. All samples were stored at -80°C before testing and were examined retrospectively in mixed batches without knowledge of the clinical diagnosis.


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  		TABLE 1. Detection of M. fermentans DNA by PCR in synovial fluids of patients with arthritis

 
Of the 17 patients diagnosed as having reactive arthritis, eight had sexually acquired reactive arthritis, two of whom were found to have Chlamydia trachomatis infection of the genital tract and nine had arthritis following gastrointestinal infection. Of the latter, Campylobacter infection was identified in two and Salmonella enteritidis in one. A microbial cause was not identified in the remaining patients. Detailed analysis of synovial fluid samples for cell counts and for the presence of microbial antigens or DNA was not carried out.

Aliquots (500 µl) of SF were diluted with an equal volume of phosphate-buffered saline and centrifuged at 13000 g for 15 min. The DNA extraction method [25] and the PCR assay [24] were as described previously. Negative controls were used as follows. During DNA extraction, a tube containing only lysis buffer was included after every five samples. When preparing samples for the PCR, an additional tube containing water instead of an aliquot of sample was added after every third or fourth tube and gloves were changed after each of the negative control tubes. During the preparation of the second round of the PCR, a further control tube containing water instead of amplified product from the first round was added after every 20 tubes and gloves were changed after every tube. Although laborious, this process ensured that there was no carry-over of DNA via the gloves. The primers and thermal cycling profile for this assay were as described previously [24]. Positive PCR samples, indicated by the presence of 104-base pair products, were confirmed by Southern blot analysis using the probe as described. Gels of all samples, positive or negative, were transferred by Southern blot and then hybridized with the probe to confirm positivity or negativity. In our hands this assay is able to detect 10-15 g of M. fermentans DNA, equivalent to about two mycoplasma genomes per reaction, and is 100–1000 times more sensitive than culture [24].


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Mycoplasma fermentans DNA was detected in 24 samples from patients with inflammatory arthritides (Table 1Go). Synovial fluid samples from six (17%) of 35 patients with RA were positive. In addition, M. fermentans DNA was detected in samples from 11 (25%) of 44 patients with undifferentiated seronegative arthritis and four (17%) of 24 patients with psoriatic arthritis. Also, samples from three (18%) of 17 patients with reactive arthritis were positive. In contrast, none of the SF samples from 19 patients with OA or seven with crystal synovitis was positive. Thus, M. fermentans DNA was detected significantly more often in the joints of idiopathic inflammatory arthritides [24 (20%) of 120] than in those of patients with degenerative or metabolic arthritis (none of 26) (P=0.008, Fisher's exact test). When patients with RA were compared with those with OA and crystal synovitis, the results were still statistically significant, i.e. six (17%) of 35 compared with none of 26 (P<0.01).

Twenty-one (17.5%) patients in the inflammatory arthritis categories had had disease for less than 1 yr. Of these 21, seven (33%) had positive results in contrast to 17 (17%) of the remaining 99 patients in these categories (P=0.09, {chi}2 test). No differences in age, male:female ratio or distribution of joint disease were demonstrable between patients positive or negative for M. fermentans DNA within an individual disease category.


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
We have used a sensitive and specific assay, with appropriate negative controls, which demonstrates the presence of M. fermentans DNA in the joints of some patients with arthritis. Positive results were found in 20% of subjects with non-metabolic inflammatory arthritis, including RA, but not at all in patients with OA or crystal synovitis. We did not detect any clinical differences between M. fermentans-positive and M. fermentans-negative patients.

The detection of M. fermentans DNA in joint material from a minority of patients with RA and other forms of inflammatory arthritis is consistent with the findings of Schaeverbeke et al. [25]. However, there is no clear consensus on these findings. Johnson et al. [27] in the UK reported the detection of M. fermentans DNA in 31 (91%) of 34 SF samples from patients with RA, nine (82%) of 11 samples from patients with other inflammatory arthritides and none of 10 samples from patients with OA. They used a universal 16S ribosomal RNA primer set which detects 10 mycoplasma species found in humans and at least 37 other mycoplasma species. The PCR product was sequenced and identified as M. fermentans. In contrast, Hoffman et al. [26] in the USA failed to detect M. fermentans DNA in any joint specimens from eight patients with RA. They had used a 16S ribosomal RNA PCR with primers which were specific for the mycoplasma genus. It is not easy to explain the discrepancy between these results. The small number of samples examined by Hoffman et al. [26] and their use of the genus-specific PCR may be relevant factors, but it seems unlikely that the explanation is a geographical one.

It is possible that antigens of non-viable or viable M. fermentans persist in the joint for weeks, months or years and stimulate chronic inflammatory changes. Viable M. fermentans organisms have been cultured from synovial samples from some French patients [20, 21, 28]. This raises the question of how long such organisms might survive. Insufficient patients were examined in the current study to be confident that the proportion of M. fermentans DNA-positive patients decreased as the duration of disease increased. A more robust approach to resolving the question of organism survival would be to take sequential synovial samples from patients with chronic arthritis, test them by a sensitive PCR assay and so determine whether M. fermentans DNA is present transiently, intermittently or persistently. Another approach would be to seek M. fermentans messenger RNA, which has a short half-life, using reverse transcription–PCR to determine whether the DNA came from viable microorganisms. If persistence were demonstrated, the effectiveness of anti-mycoplasmal antibiotic therapy in eradicating such organisms should be tested before evaluating the possible clinical benefits of such therapy.

If the finding of M. fermentans in the throat, peripheral blood mononuclear cells and urine of up to 20% of individuals attending a sexually transmitted diseases clinic without arthritis or evident mycoplasmal infection [24] can be extrapolated to the general population, it is clear that the opportunity for spread of the mycoplasma to joints exists for a large number of people. The presence of synovitis might favour the secondary deposition of microorganisms within inflamed tissue. Currently, the question of whether such intra-articular bacteria might have a primary aetiological role, a secondary enhancing and/or perpetuating role or no biological effect at all is unanswered. However, if approximately 20% of cases of certain forms of inflammatory arthritis are associated with the presence of M. fermentans in the joint, it is pertinent to ask and seek to answer this question. Potential criteria for defining a causal role have been put forward [29] and attempts should be made to determine whether it is possible to fulfil these. However unlikely the involvement of mycoplasmas in the causation or perpetuation of arthritis may seem, this avenue remains worthy of further exploration.


   Notes
 
Correspondence to: A. Keat. Back

* Present address: Department of Microbiology, Eastman Dental Institute, 256 Grays' Inn Road, London WC1X 8LD, UK. Back


   References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Submitted 10 July 2000; Accepted 4 June 2001


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