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Clinical and Diagnostic Laboratory Immunology, July 2004, p. 789-791, Vol. 11, No. 4
1071-412X/04/$08.00+0     DOI: 10.1128/CDLI.11.4.789-791.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Impact of Serological Methodology on Assessment of the Link between Chlamydia pneumoniae and Vascular Diseases

Boulos Maraha,1* Martin den Heijer,2 Jan Kluytmans,3 and Marcel Peeters4

Regional Laboratory for Medical Microbiology, Dordrecht,1 Department of Endocrinology and Department of Epidemiology and Biostatistics, University Medical Center, Nijmegen,2 Department of Clinical Microbiology, Amphia Hospital, Breda,3 Department of Clinical Microbiology, St. Elisabeth Hospital, Tilburg, The Netherlands4

Received 18 November 2003/ Returned for modification 13 January 2004/ Accepted 24 March 2004


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ABSTRACT
 
We assessed the impact of five serologic tests on the link between Chlamydia pneumoniae and abdominal aortic aneurysms (AAA). The results of the tests were inconsistent. Agreement among the five tests was generally poor. Detection of the link between C. pneumoniae and AAA depends on the serologic methodology chosen.


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TEXT
 
Seroepidemiologic studies that investigated the link between Chlamydia pneumoniae and vascular diseases have reported inconsistent results, ranging from a strong link to no link at all (3). This discrepancy might be due to methodological factors (15). Two recent studies have demonstrated that the detection of a link between C. pneumoniae and coronary artery disease depends on the choice of serologic methods (6, 15). The serologic link between C. pneumoniae and vascular diseases has been assessed by microimmunofluorescence tests (MIF) and enzyme-linked immunosorbent assays (ELISA). However, these serologic tests lack sufficient reliability and standardization (4). A poor agreement among the results of these tests has been reported (6, 13, 15).

In this case-control study, we investigated whether the choice of serologic tests influences the detection of a link between C. pneumoniae and abdominal aortic aneurysms (AAA). Moreover, we evaluated the agreement among the results of these tests.

The study population used was previously described (9). Patients with AAA and healthy controls were included and matched by age and sex. Serum samples were tested for the presence of C. pneumoniae immunoglobulin G (IgG) antibodies by five serologic tests, i.e., the Chlamydia MIF IgG (MRL Diagnostics, Cypress, Calif.), the Chlamydia IgG SeroFIA (Savyon Diagnostics Ltd., Ashdod, Israel; Savyon MIF), the Chlamydia IgG rELISA (Medac Diagnostica, Hamburg, Germany), the SeroCP IgG (Savyon Diagnostics Ltd.; Savyon ELISA), and the Elegance C. pneumoniae IgG ELISA (Bioclone, Marrickville, Australia).

The MRL MIF uses, as the antigen, purified C. pneumoniae (strain TW 183) elementary bodies (EB) diluted in 3% yolk sac to add contrast to the background. According to the manufacturer's product information, the EB are purified by removing the genus-specific lipopolysaccharide (LPS). The Savyon MIF and the Savyon ELISA also use purified C. pneumoniae (strain TW 183) EB as the antigen. The Medac rELISA uses a recombinant LPS fragment as the antigen. For the Bioclone ELISA, purified C. pneumoniae outer membrane protein complexes are used as the antigen. All tests were performed and interpreted in a blinded fashion by the same technician, according to the manufacturers' instructions.

We used odds ratios and a 95% confidence interval for estimating the relative risk. Kappa ({kappa}) values were used to assess the agreement among the tests. The following guidelines were used in the interpretation of {kappa}: if {kappa} was <0.2, agreement was poor; if {kappa} was 0.21 to 0.4, agreement was fair; if {kappa} was 0.41 to 0.6, agreement was moderate; if {kappa} was 0.61 to 0.8, agreement was good; and if {kappa} was 0.81 to 1.0, agreement was very good (1). P values of <0.05 were considered statistically significant.

The study population included 88 patients with AAA and 88 healthy controls. The characteristics of the study population are shown in Table 1. The results of the five tests were inconsistent (Table 2). In the patient group, seropositivity rates varied from 52% (46 of 88 patients) with the Medac rELISA to 97% (85 of 88) with the MRL MIF. In the healthy controls, a similar variation was found: 55% (48 of 88 controls) were positive with the Medac rELISA compared to 97% (85 of 88) with the Bioclone ELISA.


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  		TABLE 1. Baseline characteristics of patients with AAA and control subjectsa


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  		TABLE 2. Association between C. pneumoniae seropositivity and AAA according to five different testsa

The MRL MIF was the only test that demonstrated a significant link between C. pneumoniae and AAA. The other four tests failed to demonstrate any link. However, the MRL MIF also failed to demonstrate a link when higher IgG titers were used as cutoffs. Although the results of the two MIF used in our study were read by the same technician, there was poor agreement between the two tests. This implies that in addition to the subjective reading of MIF results, other factors may contribute to the disagreement among results of C. pneumoniae serologic tests. The test procedure, the type of antigen, the antigen's purity, and the concentration of the antigen may also account for poor agreement among the results of these serologic tests (5). The link between low titers of C. pneumoniae IgG and AAA, demonstrated by the MRL MIF, might be the result of a cross-reaction to the antigen used in the test from sources other than C. pneumoniae (8). These sources, either infectious or noninfectious, might be associated with AAA and confound the association between C. pneumoniae and AAA.

The agreement among the results obtained by the five serologic tests was generally poor (Table 3). Inter- and intralaboratory variations and a poor agreement among results of serologic tests of C. pneumoniae have also been demonstrated by others (6, 13, 15). Ranges of agreement from 59 to 90% have been reported (13, 15). Hoymans et al. (6) found poor agreement between results of the MIF and the Medac rELISA, but three other ELISA showed moderate to good agreement in results with the MIF (6).


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  		TABLE 3. Agreement of {kappa} values between the serological results of the five different tests for the patient group and the healthy controls

There is evidence that C. pneumoniae serologic tests are less specific than previously realized (5, 8, 11). Cross-reactivity between C. pneumoniae and Chlamydia species in the MIF has been demonstrated (8, 11). This is probably due to a lack of LPS removal from the EB during antigen preparation (11). It is also possible that Chlamydia-like microorganisms, Bordetella pertussis and parvovirus, cause serologic antigenic cross-reactivity with C. pneumoniae (6, 7, 10, 12, 14).

Our results support the findings of recent studies which have shown that methodology has an important impact on whether a link is found between C. pneumoniae and vascular diseases (6, 15). This indicates that methodological factors are partly responsible for the conflicting results in the literature concerning the role of C. pneumoniae in the development of vascular diseases (2, 3).

This study shows that the detection of a serologic link between C. pneumoniae and AAA depends on which test is used to measure C. pneumoniae antibodies. Further studies should focus on optimizing and standardizing C. pneumoniae serologic methods.


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ACKNOWLEDGMENTS
 
We thank M. Kerver for her technical assistance.


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FOOTNOTES
 
* Corresponding author. Mailing address: Regional Laboratory for Medical Microbiology, P.O. Box 899, 3300 AW Dordrecht, The Netherlands. Phone: 31 78 652 31 61. Fax: 31 78 652 31 20. E-mail: b.maraha{at}asz.nl. Back


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Clinical and Diagnostic Laboratory Immunology, July 2004, p. 789-791, Vol. 11, No. 4
1071-412X/04/$08.00+0     DOI: 10.1128/CDLI.11.4.789-791.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Ieven, M. M., Hoymans, V. Y. (2005). Involvement of Chlamydia pneumoniae in Atherosclerosis: More Evidence for Lack of Evidence. J. Clin. Microbiol. 43: 19-24 [Full Text]  

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