Division of Rheumatology, Department of Internal Medicine, University Hospital, Geneva, Switzerland
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Abstract |
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Methods. Immunoblotting, enzyme-linked immunosorbent assays using six synthetic peptides or recombinant antigens and a microimmunofluorescence test were used to determine the presence of IgG, IgM and IgA in serum samples from 17 patients with C. trachomatis reactive arthritis. Twenty patients with other inflammatory arthropathies without evidence of urogenital C. trachomatis infection were used as controls.
Results. The best association of sensitivity (76%) and specificity (85%) was obtained when IgG and/or IgA reactivity to two species-specific antigens was determined. These antigens were synthetic peptides, derived from species-specific epitopes in the variable domain IV of the major outer membrane protein (MOMP) (Labsystems, Finland) and recombinant polypeptide encoded by open reading frame 3 of the plasmid (pgp3).
Conclusions. IgG and/or IgA anti-MOMP-derived peptides and anti-pgp3 could be useful for the diagnosis of probable C. trachomatis reactive arthritis.
KEY WORDS: Chlamydia trachomatis, Sexually acquired reactive arthritis, Antibodies, MOMP, Pgp3.
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Introduction |
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In two previous studies, we compared serum anti-Chlamydia antibody responses, obtained for patients with well defined urethral/endocervical infection, with those obtained for healthy blood donors. The best sensitivity (86%) with a specificity of 81% was obtained for immunoblot results, when the number of individuals with 10 IgG and/or
2 IgM responses to the different C. trachomatis antigens was considered [2]. Concerning antibody responses to peptides or recombinant antigens, the best sensitivity (79%) associated with the best specificity (82%) was obtained when IgG responses to both synthetic peptides, derived from species-specific epitopes in the variable domain IV of the major outer membrane protein (MOMP) (Labsystems, Finland) and recombinant polypeptide encoded by open reading frame 3 of the plasmid (pgp3) were considered [3]. MOMP is a surface-exposed, integral membrane protein of approximately 40 kDa, found in both the extracellular infectious elementary bodies and the non-infectious intracellular reticulate bodies. Pgp3 is a protein of approximately 27 kDa, predominantly found in chlamydial outer membrane complex preparation [4].
Therefore, we evaluated whether these new assays can also increase sensitivity and specificity of the serological diagnosis of C. trachomatis reactive arthritis. The specificity was evaluated from samples of patients with inflammatory arthropathies without evidence of C. trachomatis infection, half of the cases being other bacterial arthropathies. Such controls are necessary in view of unspecific reactions possibly due to inflammation and/or presence of cross-reacting antibodies, due to other bacterial infections.
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Patients and methods |
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Immunoblot analyses of sera
They were performed as previously described [5].
Recombinant protein preparations and measurement of antibodies to hsp70, OMP2, hsp60 and pgp3 by enzyme-linked immunosorbent assays (ELISA), measurement of antibodies to C. pneumoniae, MOMP-derived peptides and LPS by commercially available ELISA and microimmunofluorescent (MIF) tests
Experimental conditions were previously described [2, 3].
Calculations
Sensitivity, specificity and agreement were calculated as described previously [6].
Statistical analysis
Where appropriate, results were analysed by the chi-square test.
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Results |
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The MIF test was used to determine the presence of anti-C. trachomatis antibodies in seven samples from patients with C. trachomatis reactive arthritis. For IgG measurement, 14% were found to be positive whereas 71% of these samples had IgG antibodies to the MOMP-derived peptides and pgp3 (P=0.031). For IgA, no sample was found to be positive with MIF test whereas 29% had IgA antibodies to the MOMP-derived peptides and pgp3 (not significant) (data not shown).
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Discussion |
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In a previous study, comparing acute C. trachomatis infected patients with healthy blood donors, we obtained the best results with immunoblots, when the number of individuals with 10 IgG and/or
2 IgM responses was considered (sensitivity, 86%; specificity, 81%) [2]. In the present study, using as controls, patients with inflammatory arthropathies unrelated to C. trachomatis, immunoblot results were found to be less sensitive (69%) and specific (75%). This lower sensitivity should be related to the observation we made earlier that significantly fewer Chlamydia antigens were recognized on the blots, by sera from patients with reactive arthritis than by those with uncomplicated C. trachomatis genito-urinary infection [5]. The lower specificity obtained in this study, with samples from patients with inflammatory arthropathies, compared with the one obtained with samples from healthy blood donors [2] suggests that some antibodies might be elicited by other micro-organisms, as 50% of these control patients had another bacterial infection. This hypothesis is also supported by low specificities obtained for IgG binding to other antigens tested in ELISA: LPS (30%), OMP2 (45%) and hsp60 (55%). As LPS and OMP2 are genus-specific antigens, low specificities obtained with them can be attributed to cross-reactivity with C. pneumoniae as 42% of these patients had IgG antibodies to it. If cross-reactivity to C. pneumoniae can also be involved for hsp60 recognition [3], cross-reactivity to other bacterial hsp60 is also possible because, for half of them, the arthropathy was dependent on Borrelia burgdorferi, Brucella or Staphylococcus aureus.
The most appropriate tests for the serodiagnosis of patients with C. trachomatis reactive arthritis were found to be IgG and/or IgA reactivity to MOMP-derived peptides + pgp3. Compared with a previous study [1], a lower sensitivity was obtained for MOMP-derived peptide antibodies, due to a modification of the cut-off calculation indicated by the manufacturer (Labsystems).
In conclusion, since C. pneumoniae infections are common and able to elicit cross-reacting antibodies, such as anti-LPS, -OMP2 or -hsp60 and since other bacterial infections, involved in inflammatory arthropathies, are also able to elicit cross-reacting antibodies such as anti-hsp60, only species-specific antigens, such as MOMP-derived peptides or pgp3, should be used. The improvement of sensitivity and specificity obtained with recombinant pgp3 prepared under native conditions (in order to retain an antigenic structure able to detect antibodies directed to conformational epitopes) is shown for the first time in the serodiagnosis of this disease. Finally, if the MIF test is the most documented serological method and is commonly accepted as the reference assay, we did not find it as the best test. ELISA using MOMP-derived peptides or pgp3 are rapid methods, completely objective, giving reproducible results, easier to perform than the MIF test and appeared to be more helpful in the serodiagnosis of C. trachomatis reactive arthritis. However, due to the difficulty of having well characterized patients with this uncommon disease, the number of samples tested was small and the results obtained with these tests should be confirmed in bigger studies.
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Acknowledgments |
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Notes |
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References |
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