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Immunocomb Chlamydia Bivalent Assay to Study Chlamydia Species- Specific Antibodies in Patients With Coronary Heart Disease

Posted on: Thursday, 21 April 2005, 03:00 CDT

Background & objectives: Serological evidences suggested an association between Chlamydia pneumoniae infection and coronary heart disease (CHD). Efficacy of available serological tests for detection of C. pneumoniae antibody has been debated. The present study was carried-out to assess the efficacy of Immunocomb Chlamydia bivalent IgG assay vis--vis micro immunofluorescence (MIF) test in detecting C. pneumoniae and C. trachomatis - specific antibodies in patients with CHD.

Methods: Serum samples collected from clinically confirmed cases of CHD (n=114) were subjected to Immunocomb Chlamydia bivalent assay and the standard MIF test. Antibodies specific to C. pneumoniae and C. trachomatis were detected quantitatively.

Results: Though Immunocomb Chlamydia bivalent test yielded 73.7 per cent positivity for C. pneumoniae- specific IgG antibody (compared to 50.8% by MIF), the specificity of Immunocomb was found only 32.14 per cent. Positive and negative predictive values of Immunocomb assay were 54.8 and 60.0 per cent respectively.

Interpretation & conclusion: The findings of the present study indicated that though Immunocomb assay was inferior to MIF, it can be used as a method for presumptive serology due to its rapidity and ease of performance. Wherever possible, one or more additional tests should also be performed to increase the specificity of such studies.

Key words Chlamydia pneumoniae * C. trachomatis * Immunocomb Chlamydia bivalent assay * coronary heart disease * microimmunofluorescence

Chlamydia pneumoniae is reported to be associated with coronary heart disease (CHD)1. Seropositivity to this organism confers some degree of increased risk of coronary events and the organism has been detected in atherosclerotic plaques. The serological evidence of association between C. pneumoniae infection and acute myocardial infarction has also been reported1. However, no definite etiological link has been established so far due to difficulty in diagnosing chronic blood vessel infection, although many studies have examined the relationship between raised C. pneumoniae antibody titres and vascular disease2. A review published in 1997 identified 18 case- control studies with 2700 cases, reported an odds ratio (OR) of 2 or more suggesting a strong association between serological markers of C. pneumoniae infection and vascular disease3. A recent meta- analysis of 16 prospective case-control studies reported a weak association between raised C. pneumoniae IgA titres and CHD and a non-significant association between raised IgG titres and CHD4. As majority of patients with CHD do not undergo surgery, serology for detection of raised levels of C.pneunioniae antibodies has been used as indirect measure to assess the association of C. pneumoniae with CHD5-7.

Persistently raised IgG and IgA titres, determined by micro immimofluorescence (MIF) or a variety of enzyme immunoassays (EIAs), have been considered as one of the criteria for chronic infection. However, there are no uniform cut-off titres to define seropositivity. and it is unclear whether seropositivity reflects chronic, or merely past, infection8. Though the MIF test is technically challenging, prone to subjective variations compared to more commonly used EIAs, it is considered as the gold standard for detection of Chlamydia antibody9.

Many factors influence selection of appropriate diagnostic test for serological detection of chlamydial antibodies. These include number of samples to be processed, cost of the test, and expertise available. Further, before using a new test, sensitivity and specificity of each assay needs to be considered10. Keeping this in view, in the present study we used Immunocomb Chlamydia bivalent assay and compared the efficacy of this test with MIF to detect C. pneumoniae and C. trachomalis specific IgG antibodies in patients with coronary heart disease.

Material & Methods

Study samples: A total of 114 serum samples from clinically confirmed cases of stable coronary heart diseases (85 males, 29 females) were included in the study. The mean age was 55.1212.5 yr (age range was 45-75 yr). All samples were collected from the consecutive selected patients attending cardiology clinic of All India Institute of Medical Sciences, New Delhi from June 2001 to June 2002.

Immunocomb test: Commercial Immunocomb Chlamydia Bivalent IgG kit (Organics, Israel) was used as per the instructions of the manufacturer. The values were recorded as the approximate titre of C. pneumoniae IgG antibodies. The positive control provided with the kit was tested with each batch using the same procedure. Also a known Chlamydia antibody positive serum in the microimmuno fluorescence assay was tested using 3 dilutions viz., 1:16, 1:64 and 1:128 and the colour intensity produced were noted for comparison.

Microimmunofluorescence assay: MIF test11 was used to detect Chlamydia species-specific antibodies in serum samples with the kit provided by IO International Ltd, UK. Briefly, each antigen slide provided with the kit consisted of an 18-well teflon-coated glass slide containing antigen dots. Each well was pre-coated with 5 dots of antigen, each consisting of approximately 106 elementary bodies of C. lrachomatis serovars A-C (pooled), C. trachomatis serovars D- K (pooled). C. trachomatis serovars L^sub 1^-L^sub 3^ (pooled), C. psittaci and C. pneumoniae grown in yolk sacs of embryonated hen's eggs. There was a separate dot of sonicated uninfected yolk sac membrane as control in each well of the slide. The screening of the serum samples was done in three serum dilutions i.e., 1:16, 1:64, 1:128 in phosphate buffer saline (PBS) pH 7.4 and any serum showing positivity in 1:128 dilution was tested in further dilutions. With each batch of the test, positive and negative control sera (provided with the kit) were included. In the positive specimens, chlamydia elementary bodies were observed as bright fluorescent regular round apple green particle. Any serum sample giving a positive result in 1:16 dilution was considered as positive and the highest dilutions of the serum giving results was taken as the antibody titre of that serum sample.

Statistical analysis: Agreement analysis was done using sensitivity, specificity, positive and negative predictive values and diagnostic accuracy of the test.

Results

In both serological assays (Immunocomb and MIF), samples giving a titre of 1:16 or more were considered positive. About 47 per cent samples showed positivity for both C. pneumoniae and C. trachomatis antibodies in MIF compared to 36 per cent by Immunocomb (Table I). The results for quantitative antibody detection against C. pneumoniae and C. trachomatis using both the assays are given in Table II. Using MIF assay, 58 (50.8%) serum samples were positive for C. pneumoniae and 45 (39.5%) were positive for C. trachomatis antibodies. In the immunocomb assay, 84 (73.7%) specimens were positive for C. pneumoniae antibody; whereas 21 (18.4%) were positive for C. trachomalis antibody (Table II).

Sensitivity and specificity of the Immunocomb assay was 79.3 and 32.14 per cent in detecting C. pneumoniae antibodies (Table III), and 15.6 and 79.7 per cent respectively in detecting C. trachomatis antibodies, when compared to MIF (Table IV). Positive predictive value of Immunocomb for C. pneumoniae was 54.8 per cent and negative predictive value was 60.0 per cent (Table III); while positive and negative predictive values for C. trachomatis were 33.3 and 59.0 per cent respectively. The diagnostic accuracy of Immunocomb test for C. pneumoniae was 56.1 per cent and for C. trachomatis 54.4 per cent.

Table I. Chlamydia pneumoniae and C tracliomatis antibody positivity in patients with coronary heart disease (n=114)

Table II. Chlamydia pneiimoniae and C. trackomatis IgG titres in patients with coronary heart disease (n=114)

Table III. Results of Chlamydiapneumonias antibody detection by microimmunofluorescence and Immunocomb assays

Table IV. The results of Chlamydia trachomatis antibody detection by microimmunofluorescence and Immunocomb assays

Discussion

The prospective meta-analysis, case-control and epidemiological studies have demonstrated association between elevated levels of C. pneumonias antibody titre and myocardial infarction (Ml) or chronic CHD. While presence of elevated antibody titre is generally considered indicative of past chronic infection, but no clear information is available about the time span of persistence of chlamydial antibodies in individuals after single episode of infection11,12.

In the present study 73.7 and 50.8 per cent patients with CHD showed serologic evidence of C. pneunwniae infection by Immunocomb and MIF assays respectively. The Immunocomb test was used to examine a panel of sera for which clinical details and serological results were available.

Detection of species-specific chlamydia antibody by MIF is very sensitive, specific and is considered as gold standard. It has been widely used in the diagnosis of C pneumonias infections. In an earlier study, Immunocomb test results were shown to correlate well with MIF13. MIF requires a high level of expertise for correct interpretation, and also an expensive fluorescent microscope. On the other hand, Immunocomb assay is easy to perform and interpretation of results is comparatively less subjective.

Since th\e IgG positivity against C. pneumoniae amounts for more than 50 per cent in healthy population, cross-reactivity with other chlamydial species may lead to unacceptably high ratio of false positives in patient groups with other prevalent chlamydial infection(s). In a multivariate analysis in USA, of the 246 incident cases of CHD 65 per cent had IgG antibody titre for C. pneumoniae compared with 55 per cent of healthy cases14. In a study on serological screening on healthy blood donors in Delhi, C. pneumoniae antibodies were found in 42.77 per cent blood donors by MIF15. Serological evidence of past C pneumoniae infection was also reported in up to 20 per cent in UK population16. In earlier study on 266 CHD patients we had shown 41.3 per cent seropositivity using MIF, compared to 30 per cent of age and sex matched control17. Therefore, need for a species-specific test is often felt in larger community-based studies to assess association between infection with C. pneumoniae and CHD.

Comparison of different assays for large-scale clinical trials may be valuable, but results should be viewed with caution. It is possible to make any assay appear sensitive by comparing it with a sub-optimal test, or by comparing the results of specimens of newer, more specific assays18,19. Though several tests that detect chlamydial antibody by EIA (Immunocomb) are commercially available, MIF is the only serological test that detects species and serovar- specific responses against genus Chlamydia20. MIF and Immunocomb tests also, detect reactivity to genus-specific antigen of chlamydial elementary or reticulate bodies21.

Unlike C. pneumoniae, role of C. trachomatis in coronary heart disease is not clear. Since Immunocomb assay employs lipopolysaccharide (LPS) extracted from C. trachomatis L2 and LPS extracted from C. pneumoniae elementary bodies on two separate antigenic spots, interpretation of the results was crucial.

Our findings indicated that Immunocomb assay was inferior to MlF in detecting C. pneumoniae antibodies in serum samples. Therefore, Immunocomb can only be used as a presumptive test; confirmatory diagnosis must include combination of tests, rather than deriving inference on the basis of single test results.

Acknowledgment

Authors acknowledge the financial support from the Indian Council of Medical Research. New Delhi.

References

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Gita Satpathy, Anjana Sharma & Suman Vasisht*

Ocular Microbiology section, Dr R.P. Centre for Ophthalmic Sciences, *Department of Cardiology, C.N. Centre, All India Institute of Medical Sciences, New Delhi, India

Received February 16, 2004

Reprint requests: Dr Cita Satpathy, Additional Professor, Ocular Microbiology section, Dr R.P. Centre for Ophthalmic Sciences All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India

e-mail: gita.satpathy@gmail.com

Copyright Indian Council of Medical Research Mar 2005

Source: Indian Journal of Medical Research

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