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J Clin Microbiol. Sep 2003; 41(9): 4049–4053.
PMCID: PMC193860

Importance of Methodology in Determination of Chlamydia pneumoniae Seropositivity in Healthy Subjects and in Patients with Coronary Atherosclerosis

Abstract

Enzyme immunoassays (EIAs) for the detection of Chlamydia pneumoniae antibodies were compared to the microimmunofluorescence (MIF) test, the reference method. Furthermore, we assessed the hypothesis that a possible relationship between Chlamydia pneumoniae immunoglobulin G (IgG) antibodies and coronary artery disease is dependent on the type of EIA. Sera from 112 healthy men (mean age, 50.1 years) were tested for antibodies against Chlamydia pneumoniae by five commercial test kits: Focus Chlamydia MIF IgG test, Labsystems Chlamydia pneumoniae IgG EIA (LS EIA), R-Biopharm Elegance Chlamydia pneumoniae IgG EIA (RB EIA), Medac Chlamydia pneumoniae IgG sandwich enzyme-linked immunosorbent assay ELISA (MCp sELISA) and Medac Chlamydia IgG recombinant enzyme-linked immunosorbent assay ELISA (MC rELISA). Sera from 106 consecutive male patients (mean age, 63.6 years) undergoing diagnostic coronary angiography were also examined using the Focus MIF, LS EIA, MCp sELISA, and MC rELISA techniques. The agreement between LS EIA (65 to 83% [controls-patients]) or MC rELISA (49 to 61%) and Focus MIF (78 to 83%) was average to fair (κ = 0.597 and 0.234, respectively). MCp sELISA and RB EIA showed good agreement with MIF (κ = 0.686 and 0.665, respectively), with 80 to 89 and 79% of individuals reacting positively. A significant difference in seroprevalence between patients and healthy subjects was observed with the LS EIA, while seropositivities in the two study groups appeared equal when the Focus MIF assay was applied. The MC rELISA and MCp sELISA gave statistically significant differences in antibody seroprevalence in patients with two-vessel disease or when the patient group combined individuals with a two- or a three-vessel disease, respectively. The concordance between MIF and other commonly used serological assays for C. pneumoniae IgG antibody detection is good to fair. The choice of serological assay has important implications for C. pneumoniae antibody seroprevalence, as well as for the relationship between C. pneumoniae seropositivity and coronary artery disease.

In 1988, a provocative relationship between positive serology for Chlamydia pneumoniae and atherosclerosis was reported by researchers from Finland. Saikku et al. (23) found that 68% of patients with acute myocardial infarction and 50% of patients with chronic coronary heart disease (CHD) had raised immunoglobulin G (IgG) (≥1:128) and IgA (≥1:32) titers against C. pneumoniae measured with the microimmunofluorescence (MIF) test compared to only 17% of the controls. In addition, nearly 70% of 38 patients with acute myocardial infarction also showed significant seroconversion against chlamydial lipopolysaccharide (LPS). In the Helsinki Heart Study (24), these authors demonstrated that chronic C. pneumoniae infection—which they defined by the presence of an elevated IgA titer and LPS containing immune complexes—was an independent risk factor for the development of CHD 3 to 6 months before the coronary event. Since these initial reports, many studies have confirmed an association between antibodies to C. pneumoniae and coronary artery disease, but negative findings have also been frequently reported (2, 4, 12, 13, 18, 19).

The MIF test is considered to be the international reference “gold standard” for determination of Chlamydia seropositivity (7). The test allows the simultaneous detection of antibodies to all three Chlamydia species that can be found in humans and is able to differentiate among the IgG, IgA, and IgM antibody classes. However, it has some major drawbacks: it is time-consuming and requires skilled personnel for interpretation of the slides. Furthermore, the specificity of MIF has been questioned, as cross-reactions among the major outer membrane proteins of different Chlamydia species were reported (14, 28). Because of all these problems related to MIF, enzyme-linked immunosorbent assays (ELISAs) and enzyme immunoassays (EIAs) were commercially developed; they are relatively simple to perform, are less time-consuming, are more objective because of the photometrical reading of the results, and are easier to standardize, as these results are expressed in international units. Hence, different serological assays have been applied in seroepidemiologic studies investigating the association between C. pneumoniae and CHD. The use of different assays would be no problem if the agreement between the tests is high.

Therefore, we evaluated three different commonly used commercially available EIAs and ELISAs for the detection of specific IgG to C. pneumoniae, as well as a genus-specific recombinant ELISA (rELISA), in comparison with a commercially available MIF test within a healthy and a patient population and assessed the hypothesis associating C. pneumoniae IgG and ischemic heart disease.

MATERIALS AND METHODS

Study population.

Two groups of subjects were tested: group 1 consisted of 112 healthy men, with a mean (± standard deviation) age of 50.1 ± 5.4 years (range, 36 to 58 years) and with no antecedents of angina, acute myocardial infarction, coronary artery bypass grafting, coronary angioplasty, or prominent Q/QS waves on their resting electrocardiograms. Group 2 consisted of 106 consecutive male patients with a mean age of 63.6 ± 10.9 years (range, 28 to 85 years) referred to the hospital for diagnostic coronary angiography; 8 (7.5%) of these patients had normal coronary arteries, 9 (8.5%) had diffuse noncritical coronary atherosclerosis, 27 (25.5%) had a one-vessel disease (one vessel of the coronary tree showing a diameter stenosis of ≥50%), 35 (33%) had a two-vessel disease (two vessels having a diameter stenosis of ≥50%), and 27 (25.5%) were diagnosed as having a three-vessel disease (diameter stenosis of ≥50% in all three coronary vessels). Stenosis quantification was done with a computerized quantification system (Cardiovascular Angiography Analysis System II). Because in group 1, serum was available only from healthy men, females were excluded from the patient group.

Blood samples in vacuum tubes were left to clot at room temperature. The samples were centrifuged, and the sera were stored at −70°C until they were analyzed.

The local ethics committee approved the study protocol. Informed consent was obtained from all patients and controls. The study complies with the Declaration of Helsinki.

Serology.

Chlamydia MIF IgG (Focus MIF) (Focus Technologies, Cypress, Calif.), Chlamydia pneumoniae IgG EIA (LS EIA) (Labsystems, Helsinki, Finland), Elegance Chlamydia pneumoniae IgG EIA (RB EIA) (R-Biopharm, Darmstadt, Germany), Chlamydia pneumoniae IgG sandwich ELISA (MCp sELISA) (Medac, Wedel, Germany), and Chlamydia IgG rELISA (MC rELISA) (Medac) were tested on sera from healthy individuals. Sera from patients with documented coronary artery disease were analyzed for C. pneumoniae IgG antibodies using the Focus MIF, LS EIA, MCp sELISA, and MC rELISA.

The Focus MIF uses purified formalin-fixed elementary bodies of C. pneumoniae, Chlamydia trachomatis, and Chlamydia psittaci diluted in yolk sac as antigens. The MC rELISA is based on a chemically and molecularly defined Chlamydia-specific recombinant fragment of the LPS, while the RB EIA, the MCp sELISA, and the LS EIA use highly purified C. pneumoniae-specific antigen preparations which are not further specified by the manufacturers but which are free of the cross-reactive LPS component.

The assays and calculations were performed according to the manufacturers' instructions. For Focus MIF analysis, sera were screened at a dilution of 1/16 as recommended by the manufacturer. An IgG titer of ≥1:16 was defined as positive. Evaluation of slides was done blindly and in parallel by two experienced investigators.

Statistics.

Statistical analysis of results was done using SPSS for Windows version 10.1. To assess the agreement among the different tests, we used κ (nominal scale variables) as proposed by Landis and Koch (1). Guidelines for the interpretation of κ were as follows: κ < 0.20, poor agreement; κ = 0.21 to 0.40, fair agreement; κ = 0.41 to 0.60, moderate agreement; κ = 0.61 to 0.8, good agreement; and κ = 0.81 to 1.00, very good agreement. Crosstabs with the Pearson chi-square test were used when comparing the numbers of seropositive persons in the different groups of individuals. Equivocal results for the anti-C. pneumoniae assays and gray-zone results for the anti-LPS assay were considered negative. A P value of <0.05 was considered statistically significant.

RESULTS

Seroprevalence for C. pneumoniae IgG in healthy individuals and in patients determined with different assays.

A positive reaction was found in 97 of the healthy subjects (87%) by at least one test (Table (Table1).1). Forty-four percent of the positive cases demonstrated reactions in all five tests, while 34% showed positive reactions in four different tests, 9% reacted positively in three tests, 7% reacted positively in two tests, and 5% were positive by only one test (Table (Table1).1). The seroprevalences obtained with the different tests ranged from 49 to 80%: LS EIA, 65% (73 of 112); MC rELISA, 49% (55 of 112); RB EIA, 78% (87 of 112); MCp sELISA, 80% (90 of 112); and Focus MIF, 79% (89 of 112). The sera of 106 consecutive male patients who underwent diagnostic coronary angiography were also examined using the Focus MIF assay, the MCp sELISA, and the MC rELISA. Ninety of these patients (randomly chosen) were also tested by the LS EIA. C. pneumoniae antibody seroprevalences ranged from 61 to 89%: LS EIA, 83% (75 of 90); MC rELISA, 61% (65 of 106); MCp sELISA, 89% (94 of 106); and Focus MIF, 83% (88 of 106).

TABLE 1.
Significant reactions of different tests in the healthy population

Agreement with MIF.

The agreement between Focus MIF and the genus-specific MC rELISA was fair (κ = 0.234), while the agreement between Focus MIF and the species-specific EIAs was good, except for the LS EIA (κ = 0.597). Considering MIF the gold standard, the detection of true-positive samples ranged between 62 and 97%, depending on the serological assay used. The sensitivity of the LS EIA was 87% (143 of 164), that of the RB EIA was 93% (83 of 89), and that of the MCp sELISA was 97% (172 of 177), while that of the genus-specific MC rELISA was extremely low at 62% (109 of 177). The specificities were as high as 87, 87, 71, and 73% for LS EIA, RB EIA, MCp sELISA, and MC rELISA, respectively (Table (Table22).

TABLE 2.
Agreement of different tests with MIF

Seropositivity: patients with coronary atherosclerosis versus healthy controls.

Seropositivity rates obtained within both populations, and within the different angiographically defined groups, are presented in Table Table3.3. Determination of antibody status with the LS EIA resulted in a statistically significant difference in C. pneumoniae IgG seroprevalence between patients and healthy controls. In contrast, seropositivities in the two study populations appeared equal when the Focus MIF assay was applied. Statistically significant differences in antibody seroprevalence were obtained with the MCp sELISA and MC rELISA when the patient group combined individuals with a two- or three-vessel disease and when patients had a two-vessel disease, respectively. The data are presented in Table Table33.

TABLE 3.
Comparison of C. pneumoniae IgG seropositivities between patients and controls using different EIAs

Traditional risk factors for atherosclerosis.

C. pneumoniae-seropositive status determined with either of the tests was not associated with smoking, diabetes, or hypertension. Age, body mass index, and cholesterol and HDL cholesterol levels also did not differ significantly between seropositive and seronegative individuals, except that when antibodies were measured with the LS EIA or MCp sELISA, the mean age proved to be significantly higher in the seropositive group (Pearson chi-square test and Levene's test for equality of variances; data not shown).

DISCUSSION

In this study, we compared five commercially available serological assays (three species-specific EIAs, one genus-specific rELISA, and one MIF test) for the detection of C. pneumoniae IgG antibodies in the sera of 112 healthy individuals and 106 patients and found important differences. Furthermore, we detected a significant difference in the prevalence of C. pneumoniae IgG antibodies between patients with coronary atherosclerosis and healthy controls when measured with the LS EIA, while this was not the case when antibodies were detected with the Focus MIF. The results obtained with the MCp sELISA and MC rELISA were rather diffuse.

Established cardiovascular risk factors, such as hypertension, hypercholesterolemia, diabetes mellitus, age, gender, cigarette smoking, and family history, do not completely explain all new cases of atherosclerotic disease. It has been suggested that in addition to classical risk factors, infectious agents might play a role in atherogenesis (10, 27). It is now well accepted that atherosclerosis is an inflammatory disease (22), and one of the pathogens implicated as a source of and/or as a contributory factor in the inflammatory process is C. pneumoniae. The organism has been frequently detected in atherosclerotic lesions by means of PCR, in situ hybridization, immunohistochemistry, electron microscopy, and, to a lesser extent, tissue culture (15, 16, 21), but test results were often contradictory between different laboratories and gold-standard methods for diagnostic testing were lacking. Therefore, recommendations for standardized approaches in Chlamydia-related research were recently published (9). Since C. pneumoniae is difficult to culture, serological analysis still represents a routine approach for the diagnosis of C. pneumoniae infection. However, conflicting results have been published on the relationship between serology and the detection of the organism in atherosclerotic plaques (6, 17), and Ericson et al. reported no association between C. pneumoniae seropositivity and the degree of atherosclerosis (8).

We found high antibody seroprevalences in healthy subjects ranging from 65 to 80%, depending on the serological test used. As yet, there are few data on seroprevalence rates of C. pneumoniae in Belgium; seropositivity among individuals approached those reported in Finland by Tuuminen et al. (26). A possible explanation for the high antibody prevalence is that the study population consisted of only adult males. In addition, Chlamydia-like microoganisms, e.g., Parachlamydia acanthamoebae, Simkania negevensis, and four recently discovered new strains with Chlamydia-like sequences might also have added to the high seropositivity because of possible serological antigenic cross-reactivities (3, 11, 20).

There was poor agreement between the genus-specific MC rELISA and Focus MIF (Table (Table2),2), giving quite different numbers for IgG seropositivity in the control group (49 versus 79%) and in the patient group (61 versus 83%). The MC rELISA was used for Chlamydia LPS analysis and therefore was not able to differentiate between antibodies against the different Chlamydia species, e.g., C. pneumoniae, C. trachomatis, and C. psittaci. However, discordant serological results between MIF and the MC rELISA should not necessarily be ascribed to the Chlamydia species specificity versus the genus specificity of the tests, since differences could also exist in the human immunological response, as suggested by Schumacher et al. (25). In addition, cross-reactions between the different chlamydial species have recently been reported with the MIF test, bringing into question the specificity of the reference test. The fact that the MC rELISA is based upon a small Chlamydia-specific fragment of the LPS in the outer membrane instead of the total LPS content made cross-reactivities with other gram-negative bacteria less likely. However, since we did not include serological analysis for other gram-negative bacteria in our study, cross-reactivities with the LPS components from non-Chlamydia organisms cannot be completely ruled out.

In line with the findings of Hermann et al. (9), the sensitivity of the MC rELISA was very low, and therefore the assay does not seem to be appropriate for determination of antibody seroprevalence. The overall seropositivity obtained with the LS EIA (73%) assay also differed considerably from the Focus MIF results (81%), with a κ of 0.597, indicating only moderate agreement between the two tests. The sensitivity of the LS EIA appeared to be rather low compared to those of other species-specific tests. The RB EIA and MCp sELISA, however, performed well compared to MIF.

The LS EIA detected a significant difference in C. pneumoniae antibody prevalence between the controls and the patients with coronary atherosclerosis, while specific IgG antibodies to C. pneumoniae were equal in the two study groups with the Focus MIF test. No statistically significant difference was found if the MCp sELISA or the MC rELISA was used, except when the patient group consisted of individuals with a two- or three-vessel disease or a two-vessel disease, respectively (Table (Table33).

A potential limitation of our study is that we only evaluated seropositivity to IgG and not to IgA. However, we do not believe this to be a major issue, because the great majority of cross-sectional and retrospective studies that suggested a positive association relied on IgG serology to determine exposure status (5).

In this study, we demonstrated that C. pneumoniae seroprevalence, as well as the relationship between seropositivity for C. pneumoniae and coronary artery disease, is influenced by the assay applied. Serological assays might vary greatly in sensitivity and specificity. We conclude that the choice of serological test has important implications when performing seroepidemiological studies and examining whether seropositivity to C. pneumoniae represents a cardiovascular risk factor.

Acknowledgments

This work was supported by a grant from the Fund for Scientific Research-Flanders.

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