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754 LETTERS

exacerbation, to try to explain the unquestionable efficacy of tient had high titers o f rhcumatoid factor as determined by
repeated plasmaphcresis in this patient. Unfortunately, none Rose-Waaler and latex agglutination tests. In addition, she was
has been detected, emphasizing that more studies are needed HLA-DR4 positive.
in this very interesting and clinically important area. The interferon P-lB treatment was stopped for 6
weeks in an attempt to determine whether the development of
C. Michael Ncuwclt, M D arthritis was possibly related to thc intake of intcrfcron p-1s.
University of California, Sun Fruncisco In parallel, corticosteroid treatment was initiated to control the
Stanford University arthritis. The patient was followed up weekly in consultation
Pulo Alto, CA with a neurologist. After the (,-week period, there was no
Alumeda County Medical Ceizler improvement in the arthritic symptoms; thercfore, we treated
Oukland, C A the paticnt with corticosteroids in combination with sulfasala-
Ronald A. Ashcrson, M D zine. Sulfasalazinc is known to suppress experimental auto-
University of Cape Town immune encephalomyelitis (EAE) in an animal model of E A E
Cupe Town, South Africa that has similarities to human MS (2). The arthritis became
David 1. Daikh, M D more severe during this therapy. Therefore, we stopped the
University o f Cul~forniu.Sun Francisco treatment and prescribed methotrexate in a dosage of 1.5 mg
per week orally. The arthritis improved dramatically and the
MS continues to be in remission.
1. Asherson RA, Piette J-C. The catastrophic anti-phospholipid syn-
drome 1996: acute, multi-organ failure associated with anti- A previous case-control study (150 MS patients and
phospholipid antibodies; a review of 31 patients. Lupus 1996;5: 200 controls) showed that MS exhibited a significant associa-
414-7. tion with R A and psoriasis (3). There are no published data on
2. Asherson RA, Cervera R, Piette J-C. Font J, Lie JT, Borcoglu A. the use of interferon P-lB in R A treatment or on the induction
Catastrophic anti-phospholipid syndrome: clinical and laboratory of arthritis as one of the side effects of treatment with
features of 50 patients. Medicine (Baltimore). In press. interferon P-1B. In our case, interferon P-lB was not able to
3. Asherson RA. Guidelines for the investigation of patients with a prevent the onset of RA. Contrary to the case reported by
catastrophic antiphospholipid antibody syndrome. Submitted for Jabaily and Thompson, we noted no relief of R A symptoms
publication.
during the use of interferon P-1s. We therefore argue that the
4.Belmont HM, Abramson SB, Lie JT. Pathology and pathogenesis of
vascular injury in systemic lupus erythematosus: interactions of observed remission ol R A in the previously reported case is
inflammatory cels and activated endothelium. Arthritis Rheum indeed a coincidental finding unrelated to interferon treat-
1996;39:9-22. ment.
5. Abramson SB, Wcissmann G. Complement split products and the
pathogenesis of SLE. Hosp Pract 1088;23:45-55. Saifeddin Alsalameh, M D
6. Kaye BR, Neuwclt CM, London SS, DeArmond SJ. Central ner- Bernhard Manger, M D
vous system systcmic lupus erythematosus mimicking progressive Peter Kern, M D
multifocal leucoencephalopathy. Ann Rheum Dis 199251 : 1 152-6. Joachim Kalden, M D
7. Neuwelt CM, Lacks S. Kaye BR, Ellman JB, Borenstein DG. Role University of Erlungerz-Numberg
of intravenous cyclophosphamide in the treatment of severe neu- Erlangen, Germany
ropsychiatric lupus erythematosus. Am J Med 1WS;98:32-4 1.
8. Neuwelt CM, Kaye BR. Babich JF. Role of intravenour cyclo- 1. Jabaily JA, Thompson JS. Effects of interferon p-1B in rheumatoid
phosphamide (IV-CYC) in the treatment of severe neuropsychiatric arthritis: a case report [letter]. Arthritis Rheum 1997;40:1370.
lupus erythematosus (NPSLE) 30 month follow-up 1993-June, 1995 2. Prosiegel M, Neu 1, Vogl S, Hoffmann G, Wilfcuer A, Ruhenstroth-
[abstract]. Arthritis Rheum 1995;38 Suppl Y:S346.
Bauer G. Suppression of experimental autoimmune encephalomy-
elitis by sulfasalazine. Acta Neurol Scand 1990:81:237-8.
3. Midgard R. Gronning M, Riise T, Kvale G, Nyland €€. Multiple
New onset of rheumatoid arthritis during interferon sclerosis and chronic inflaminatory diseases: a case-control study.
P-1B treatment in a patient with multiple sclerosis: Acta Neurol Scand 1996:93:322-8.
comment on the case report by Jabaily and Thompson
To the Editor: Mycoplasmas in the joints of patients with
In response to the case report presented by Jabaily and rheumatoid arthritis and other inflammatory
Thompson (l), which demonstrated a possible beneficial cffect rheumatic disorders: comment on the article by
of interferon P-1B on the course of rheumatoid arthritis (RA),
Hoffman et a1
we would like to present the case of a 35-year-old female
patient who was diagnosed with multiple sclerosis (MS) in 1988 To the Editor:
and had been receiving interferon P-1B treatment since May We read with interest the article by Hoffman et al,
199.5. At the end of 1996, she started to develop a severe describing a study to investigate a relationship between myco-
polyarthritis, which fulfilled thc American College of Rheu- plasma1 infection and the development of rheumatoid arthritis
matology (formerly, the American Rheumatism Association) (RA) or juvenile rheumatoid arthritis (JRA). While serologic
criteria for RA. Laboratory studies showed an elevation of the analysis provided evidence of exposure to Mycoplasma, an
erythrocyte sedimentation rate, C-reactive protein level, and ultrasensitive polymerase chain reaction (PCR) assay failed to
y-globulin fraction on total protein electrophoresis. The pa- detect Mycoplasma DNA in the synovial fluid or tissue of
LETTERS 755

patients with RA or JRA (1). Thcse results indicate that template added to thc synovial fluid before the extraction. The
previous mycoplasmal infection is common in R A and J R A use of this technique may be considered as a control for DNA
patients, but that mycoplasmas do not persist in the joints of amplification, but does not indicate that the DNA was well
such patients. However, in a recent study, we have demon- separated from bacterial cell components and correctly ex-
strated the presence of Mycoplasma fermentans DNA in syno- tracted from the clinical samples.
vial specimcns from patients with various inflammatory arthri- If bacterial agents, as suggested by Hoffman and
tides, including R A (2). Hoffman and colleagues suggest that coworkers, can only be passengers in thc joint, it might be
such differing results might be explained by a highcr preva- possible that different bacterial populations can be observed
lence of passive carriage of M fermentans in the French among populations from different origins, justifying our differ-
population. We would like to propose another possible expla- ing results. However, other studies have shown a high preva-
nation for the disparate findings. lence of Mfermentans in different populations than ours, i.e., in
First, it is important to remember that negativc find- the throat of both British and American patients (11,12). Clark
ings in a detection study based on a PCR assay do not firmly et al have detected the presence of mycoplasmal antigens
exclude the possible presence of bacterial D N A in the speci- (among them M ,fermentans antigens) in immune complexes
men tested. In the past, several studies have concluded that from synovial fluids of patients with R A (13), and Johnson and
Chlumydiu traclzomatis DNA was absent in synovial samples colleagues recently reported detection of M fernentans DNA
from patients with sexually acquired reactive arthritis (3,4), in synovial specimens from British patients with R A (14).
until Taylor-Robinson first reported the detection of C traclio- Moreover, the serologic analysis performed by Hoffman et a1
matis D N A in such specimens (S), results thercafter confirmed evidenced an exposure to mycoplasmas in their patients. Thus,
by others. The technique cmployed is certainly critical for the M fermentuns carriage does not seem to be limited to the
sensitivity of the PCR assay, and might explain such disparate French population.
results. We would like to emphasize the role of various Hoffman et a1 consider that, in our study, the detection
technical issues that may have been critical in the study by of M fermentans in synovial specimens from patients with
Hoffman et al. various rheumatic conditions is evidence indicating that this
The use of different DNA extraction techniques has agent was not a pathogen, but was passively carried. This is
been shown to yield large diffcrences in the sensitivity of the possible, and we have discussed this point previously (2).
PCR assay (6). In their study, Hoffman and colleagues used a However, other microorganisms, such as C trachomatis, have
commercial kit to extract the DNA from synovial fluids, and also been detected in the synovium of patients with various
they simply crushed the biopsy specimen before extraction by conditions, including reactive arthritis, unclassified arthritis,
phenol-chloroform and ethanol precipitation. In our experi- and R A (15). A possible explanation could be that different
ence, such methods have seemed sufficient for extraction of bacteria are arthritogenic, and that the different clinical fea-
bacterial D N A from a culture medium, but not from clinical tures of arthritis are the consequence of the differences in the
samples, especially biopsy specimcns. Many investigators have genetic background of the patients and the related pathway of
emphasized that if bacterial agents arc present in the synovium the immune response.
in some inflammatory arthritic conditions, this seems to occur Finally, the most important difference between our
only at very low concentrations. The loss of an important studies and the one described by Hoffman and coworkers is
proportion of bacterial D N A during the extraction procedure that we identified M fermentans in synovial specimens not only
may compromise the results of a PCR assay. by PCR assay, but also previously by culture (16). Interestingly,
The choice of primers has also becn shown to be in this first study, we were able to detect mycoplasmas by PCR
crucial in the sensitivity of a PCR assay (7). In their study, in only 2 eases, while 8 were positive by culture. The techniques
Hoffman et a1 used different primers than we did. Some of of D N A extraction and amplification were significantly modi-
them were genus specific but not species specific. Interestingly, fied before we obtained the results reported in reference 2.
other authors have failed to detect mycoplasmal DNA in Although Hoffman el a1 did not suggest that the
synovial specimens when using a PCR assay with genus-specific differences in results might b e explained by a contamination of
primers (8), and we also obtained negative results when using our samples, this possibility must also be discussed. In our
such primers with clinical spccimcns, whereas they seemed study, the synovial specimens were collected in the department
efficient in dcteeting the presence of mycoplasmas in culture of rheumatology. A portion of each sample was immediately
media (Schaeverbeke T et al: unpublished data). One impor- brought to the hospital microbiology laboratory, where viable
tant factor to explain sensitivity differences among the set of Mycoplasma strains were isolated, while the other portion was
primers used by Hoffman et a1 and those used by us is the stored until PCR studies were performed. For the PCR study,
number of copies of the template gene used for amplification. we worked in 2 different places: our university microbiology
Our primers were chosen within an insertion sequence that has laboratory and the research unit of Dr. David Taylor-Robinson
been shown to be repeated more than 10 times in the bacterial in London, where no viable mycoplasmas are manipulated.
genome (9), whereas only 1 or 2 copies of the 16s ribosomal The concordance of the results in the 2 laboratories would be
R N A gene, within which Hoffman’s primers were chosen, are very surprising had there been contamination. Furthermore,
present in mycoplasmas (10). It would have been interesting to the genomic characterization of the M fermentans strains
test Hoffman’s specimens with the same primer set used in our isolated from synovial specimens showed differences among all
study. these strains (17). Contamination by a single bacterial contam-
The use of a molecular mimic was considered by inant would have led to homogeneous results, or, in case of
Hoffman et a1 to warrant the sensitivity of their PCR assay. multiple contaminants, various Mycoplasma species would
However, the molecular mimic consisted of a purified DNA have been identified.
756 LETTERS

Basically, the presence of mycoplasmas in synovial nized human pathogen Mycoplama incognitus. Gene 1990:93:67-
spccimcns from patients with arthritis docs not mean that the 72.
organism is involved in the pathogenesis of the disease. 10. Amikam D, Glaser G, Razin S. Mycoplasmas (mollicutes) have
However, there arc numerous arguments implicating myco- low number of rRNA genes. J Bacteriol 1984;158:376-8.
11, Katseni VL. Gilroy CB, Ryail BK, Ariyoshi K, Bicniasz PD, Weber
plasmas in human arthritides, including RA (18). One of the JN, et al. Mycoplasma fermentans in individua
most important is that mycoplasmas are known to be respoii- seronegative for HI\'-I. Lancet 1993;341:271-3.
sible, in many animal species, for urogcnital infection, respira- 12. Chingbingyong MI, Hughes CV. Detection of Mycoplasma fer-
tory diseases, autoimmune phenomena, and arthritis. In hu- mentans in human aaliva with a polymerase chain reaction-based
mans, urogenital infections have been attributed t o assay. Arch Oral Biol 1~96;41:311-4.
Ureuplasrnu urealyticutn, Mycoplasmu genituliurn, and Myco- 13. Clark HW, Coker-Vann MR, Bailey JS. Brown TMP. Detection of
ylusrna horninis, atypical pneumonia is duc mainly to Myco- mycoplasmal antigens in immune coniplcxcs from rheumatoid
plusrna pneurnoniae, and induction of autoimmune phenomena arthritis synovial fluids. Ann Allergy 1988;60:394-8.
has been reported during mycoplasmal infection. The implica- 14. Johnson SM, Sidebottom DA, Bruckner FE, Collins DA. Myco-
tion of mycoplasmas in human arthritis, which has been plasmas in the synovial fluid from arthritis patients [abstract]. Br J
Rheumatol 1997;36 Suppl 1 : l Y X .
debated for a long time, remains the only difference between 15. Pando JA, Yarboro C, Ellaban A, Saaibi D, Kanik K, Villalba L,
animal and human disorders attributed to mycoplasmas. In our et al. Prevalence of Chlamydia trachomatis by PCR in the syno-
opinion, this justifies continued detection studies in humans, vium of patients with early rheumatoid arthritis [abstract]. Arthri-
although methods other than culture or PCK detection assays tis Rheum 1995;38 Suppl 9:S287.
will be necessary to define the exact role of mycoplasmas in the 16. SchaeverbekeT. Renaudin H, Clerc M, Lequen L, Vernhe, JP, de
inflammatorv rheumatic disorders. Barbcyrac B, el al. Systematic detection of mycoplaamas by culture
and polymerasc chain reaction (PCR) pi-occdures in 209 synovial
Thierry Schaevcrbeke, MD, PhD fluid samples. Rr J Rheuniatol 1997;36:3 10-4.
CCcile Bebear, MD 17. Schaeverbeke T, Clerc M. Lequen L, Charron A, B6bCar CM, de
Laurence Lequen, MD Barheyrac B, et al. Genotypic ch;iracterizetion of seven strains of
Joel Dehais, MD Mycoplasma fermentans isolated from synovial fluid of patients
Christianc BCbear, MD with arthritis. J Clin Microbiol. In press.
18. Schacvcrbeke T, Vcrnhes JP, Lequen L. Bannwarth B, BCbear C ,
H6pitul Pdlegrin
Dehais J. Mycoplasmas and arthritis. Rev Rhum Engl Ed lY97;64:
Universitk Bordeaux I1 120-8.
Bordeaux. France

1. Hoffman RW, O'Sullivan FX, Schafermeyer KR, Moore TL,


Roussell D, Watson-McKown R. et al. Mycoplasma infection and To the Editor:
rheumatoid arthritis: analysis of their relationship using immuno-
blotting and an ultrasensitive polymerase chain reaction detection
We agree with Dr. Schaevcrbcke et al that mycoplas-
method. Arthritis Rheum 1997;40:1219-28. mas are of great interest as potential etiologic agents in RA. As
2. Schaeverbeke T, Gilroy CB, BCbCar C , Dehais J, Taylor-Robinson reported in our article, serologic evidence from our study
D. Detection of Mycoplasma fermentans, but not M pcnetrans, by showed that prcvious infection with M fi'imentuiis and M
PCR assays in synovial samples from patients with rheumatoid horninis appears common in the normal population, as well as
arthritis and other rheumatic disorders. J Clin Pathol 11196:49: among patients with RA and JRA. Our results using an
824-8. ultrasensitive PCR-based mcthod provided evidence, however,
3. Wordsworth BP, Hughes KA, Allan I, Keat AC, Bell JI. Chla- against the hypothesis that chronic local Mycoplasrna infection
mydia DNA is absent from the joint of patients with sexually is the etiology of KA or JRA. The central point of the letter by
acquired reactive arthritis. Br J Rheumatol 1990;29:208-10.
Schaeverb'eke and colleagues is that our findings differ from
4.Poole ES, Highton J. Wilkins RJ. Lamont IL. A search for
Chlamydia trachomatis in synovial fluids from patients with rcac- the results of their own studies, in which they found evidence
tive arthritis using the polymerase chain reaction and antigen of M fermentans in synovial fluid or tissue in 15 of 154 speci-
detection methods. Br J Kheumatol 1992;31:31-4. mens, including 8 of 38 with RA, 2 of 10 with spondylarthro-
3. Taylor-Robinson D, Gilroy CB, Thomas BJ. Kcat ACS. Detection pathy, 1 of 5 with psoriatic arthritis, and 4 o f 31 with unclas-
of Chlamydia trachomatis DNA in joints of reactive arthritis sified inflammatory arthritis (1). They enumeratc a series of
patients by polymerase chain reaction. Lancet 1c)92;34O:X 1-2. comments about the methods uscd in our study and by that,
6. Nietfeld I,, Kuipers JG, Hopf S, Zcidler H, Hammer M. Compar- challenge the conclusions of our work. We appreciate the
ison of different sample preparations of synovial lluid for detection opportunity to respond to their criticisms.
of Chlamydia trachomatis DNA hy PCR [abstract]. At-thritis
I n the design and performance of our study, we paid
Kheum l096;.39 Suppl 9:SISS.
7 . tle 0 , Marjaniiki M, Soini El, Mertsola J . Viljanen MK. Primers
great attcntion to the technical details of the experiments
ive f o r miaitivity o f PCR. Biotechniques 1004: 17:82-7. performed, and we invested considcrable time and effort in
8. PuCchal X. Roulland-Duasoix Ll. Menkhs CJ. Rheumatoid ai-thri- devcloping the methods reportcd. Because of constraints o f
tis: failure to isolate or detect Mycoplasrnas [letter]. J Rheuniatol space, we could not describe every aspcct of the development
lY07:24: 1445-6. o f the methods, nor would that have bccn appropriate. The
0. Hu WS, Wang RY-U, Liou K-S. Shih JW-K, Lo S-C. Identification esscntial features of all of our methods arc described in our
of an inscrlion-sequence-like genetic clement in (he newly rccog- article.

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