Isolation of Chlamydia pneumoniae from the Coronary Artery of a Patient with Coronary Atherosclerosis

Methods

Patients

During a 9-month period, all patients who sought heart transplantation at the Jewish Hospital Heart and Lung Institute in Louisville, Kentucky, were asked to participate in this study. Twelve consecutive patients were enrolled after informed consent was obtained.

Specimen Collection

Coronary arteries were taken from explanted hearts and were dissected in the operating room under sterile conditions. Coronary artery segments approximately 5 mm in length were placed in the following transport media: 2-sucrose-phosphate for culture of C. pneumoniae, 1 x polymerase chain reaction (PCR) buffer, and Karnofsky glutaraldehyde-paraformaldehyde. Transport vials were sealed and opened only at their final destination. Paraffin-embedded blocks were prepared in the pathology department of the Jewish Hospital Heart and Lung Institute. All specimens were taken from the operating room to the infectious diseases laboratory at the University of Louisville, where they were packaged and transported to collaborating laboratories. One serum specimen was collected from each patient before surgery. Assays were done on experimental specimens with positive and negative control specimens.

Analysis of Specimens

Culture

Processed coronary artery tissue was cultured at three laboratories. Tissue was homogenized in a sterile tissue grinder, and the resultant homogenate was applied to HEp-2 cell monolayers contained in 1-dram shell vials or 96-well microtiter plates. Culture conditions are described elsewhere [6].

Polymerase Chain Reaction

Four laboratories used PCR assays to analyze coronary artery tissue for DNA sequences specific for C. pneumoniae. Two laboratories used primers that amplify a 463-base pair fragment of the 16S ribosomal RNA gene [7]. One of these two laboratories detected amplified products by using polyacrylamide gel electrophoresis, ethidium bromide staining, and ultraviolet transillumination; the other used hybridization with an RNA biotin-labeled probe followed by an enzyme immunoassay for amplicon detection [8]. The third laboratory used primers that amplify a 437-base pair DNA target [9]. The fourth laboratory used primers that amplify the omp1 gene that encodes the major outer membrane protein of C. pneumoniae[10, 11].

Immunocytochemistry

Immunocytochemistry was done on slides prepared from paraffin-embedded tissue blocks at two laboratories using the avidin-biotin complex immunoperoxidase method, as described elsewhere [12]. The chlamydia-specific and C. pneumoniae-specific antibodies used in these assays were supplied by the Washington Research Foundation (Seattle, Washington). Tissue sections were examined by light microscopy for heavily stained areas suggestive of the presence of C. pneumoniae.

Transmission Electron Microscopy

Transmission electron microscopy was done on Epon-embedded coronary artery tissue in one laboratory using methods described elsewhere [4]. Tissue sections were examined for bacterial structures that were compatible with Chlamydia organisms.

In Situ Hybridization

One laboratory did in situ hybridization assays to test for evidence of C. pneumoniae in coronary artery tissue using methods described elsewhere [13]. In brief, an S^35-labeled DNA probe (1308 base pairs in length) that contained sequences specific for a portion of the 16S-ribosomal RNA gene of C. pneumoniae was prepared. Slides prepared from paraffin-embedded blocks were immersed in the probe and allowed to hybridize. Detection of a probe that contained radioactive DNA within the tissue sample was done with emulsion autoradiography and hematoxylin-eosin and Giemsa staining followed by examination under light microscopy at 400 x original magnification [13].

Gene Sequencing of omp1

Cultures that were positive for C. pneumoniae were confirmed by sequencing of the VS-IV domain of the omp1 gene, as described elsewhere [14]. This gene encodes the major outer membrane protein of C. pneumoniae. The VS-IV region of the omp1 gene was amplified by PCR assay, and the sequence was determined using an automated DNA sequencer (Applied Biosystems, Foster City, California).

Serologic Testing

Titers of IgG, IgM, and IgA to C. pneumoniae were determined at two laboratories by indirect immunofluorescent antibody assay, as described elsewhere [15].

Data Collection

Participating study sites sent the final results of assays to the project director at the University of Louisville. All investigators other than the project director were blinded to the presence of coronary atherosclerosis in all patients. Participating investigators were not informed of results at other study sites during the study.

Results

Patients

Twelve patients who were having heart transplantation were enrolled in this study. Coronary atherosclerosis was present on histologic examination in 10 patients (Table 1). None of the 12 patients had a clinical history of recent infection of the respiratory tract.

Isolation of Chlamydia pneumoniae

An inclusion-forming organism that reacted with a chlamydia-specific antibody was isolated from the coronary artery tissue of patient 3 at all three of the study sites that received specimens for culture (Table 1). Patient 3 was a 56-year-old man with severe coronary artery disease. The isolate from this patient (designated strain A03) was confirmed as C. pneumoniae by three methods. First, the isolate was typed as C. pneumoniae by reaction with a panel of antibodies specific for C. pneumoniae and C. trachomatis. The isolate reacted with a chlamydia-specific antibody and a C. pneumoniae-specific antibody. No reaction occurred with the C. trachomatis-specific antibody, which confirmed that strain A03 was C. pneumoniae. Second, analysis by transmission electron microscopy of strain A03 inclusions in HEp-2 cell monolayers showed characteristic pear-shaped elementary bodies. Third, sequencing of the VS-IV domain of the omp1 gene indicated complete homology with published sequences of 13 strains of C. pneumoniae[14].

Detection of Chlamydia pneumoniae

In patient 3, results of in situ hybridization showed a heavily stained area in the intimal layer of the coronary artery wall that corresponded to the area of atherosclerosis (Figure 1). Transmission electron microscopy of the coronary artery tissue in this patient also showed the presence of pear-shaped elementary bodies that were compatible with C. pneumoniae (data not shown).

Chlamydia pneumoniae was detected by at least one method in the coronary artery tissue of 7 of the 10 patients who had evidence of atherosclerosis. It was not detected by any method in the 2 patients who had no evidence of coronary atherosclerosis (Table 1).

Serologic Testing

On the basis of current criteria for serologic diagnosis [16], at least one of the two study sites detected acute antibody titers in patients 3, 6, and 8 and convalescent titers in all 12 patients. No correlation was found between antibody titers and evidence of C. pneumoniae.

Discussion

This study provides direct evidence to show the presence of viable C. pneumoniae in the atheroma of a patient with severe coronary atherosclerosis. Chlamydia pneumoniae was also seen in the atheroma of this patient by PCR assay, immunocytochemistry, electron microscopy, and in situ hybridization. These findings may indicate a local, chronic C. pneumoniae infection of the coronary arteries.

The prevalent view of the pathogenesis of atherosclerosis is the so-called response-to-injury hypothesis [1]. Persistent C. pneumoniae infection of the coronary arteries may contribute to the pathogenesis of atherosclerosis by eliciting this local response. The cellular components that are important to the pathogenesis of atherosclerosis include endothelial cells, smooth-muscle cells, and macrophages. Recent in vitro studies [17] show that C. pneumoniae is able to infect and reproduce in human smooth-muscle cells, endothelial cells of the coronary artery, and macrophages.

According to the response-to-injury hypothesis, lipid-laden smooth-muscle cells and macrophages (foam cells) migrate to and proliferate in the intimal layer of the arterial wall after an initial functional alteration of the endothelial cells of the coronary artery [1]. These cells and the extracellular matrix form a fibrous plaque that protrudes into the lumen of the artery. In 3 patients in our study, C. pneumoniae was detected in the intimal layer of the coronary artery in association with foam cells. Recent samples of the coronary artery from 49 patients 15 to 34 years of age with and without atherosclerosis were examined for C. pneumoniae by PCR assay and immunocytochemistry [18]. The organism was detected in patients with atheromatous plaques or intimal thickening but not in patients who had no evidence of atherosclerosis.

Because several laboratories participated in this study, the samples submitted to each laboratory were taken from different coronary arteries and different sections of the atherosclerotic lesions in each patient. This may explain some of the discrepancies in detection of the organism among laboratories. Because the sensitivity and specificity of the nonculture tests used in this study are not known, these discrepancies may also represent false-positive or false-negative results. Lack of standardization of current diagnostic methods may partly explain the wide range of detection of C. pneumoniae in patients with coronary artery disease. Chlamydia pneumoniae was detected in 20 of 38 patients in Seattle, Washington [19] but in only 1 of 50 patients in Brooklyn, New York [20]. It is also possible that certain unknown virulence factors or adhesion molecules of C. pneumoniae that favor coronary artery infection may be geographically distributed.

A causal relation between C. pneumoniae and atherosclerotic plaque formation will have to be shown by further investigation. If infection with C. pneumoniae is added to the list of risk factors for coronary artery disease, prevention or treatment of this infection may be an important measure in the prevention of atherosclerosis, which is the main cause of death in the United States and western Europe.

Appendix

The following are members of the Chlamydia pneumoniae/Atherosclerosis Study Group. University of Louisville, Louisville, Kentucky: Sunket Ahkee, MD, and James T. Summersgill, PhD; Jewish Hospital, Louisville, Kentucky: Brian L. Ganzel, MD, and Lynn L. Ogden, MD; The Johns Hopkins University School of Medicine, Baltimore, Maryland: Thomas C. Quinn, MD, Charlotte A. Gaydos, DPH, and Linda L. Bobo, PhD; State University of New York, Brooklyn, Brooklyn, New York: Margaret R. Hammerschlag, MD, and Patricia M. Roblin, MS; Citation Clinical Laboratories/Providence Hospital, Southfield, Michigan: William LeBar, MS; University of Washington, Seattle, Washington: J. Thomas Grayston, MD, Cho-chou Kuo, MD, PhD, Lee Ann Campbell, PhD, and Dorothy L. Patton, PhD; University of California, San Francisco, San Francisco, California: Deborah Dean, MD, and Julius Schachter, PhD.