Failure to Detect Chlamydia pneumoniae in Coronary Atheromas of Patients Undergoing Atherectomy

Steven M. Weiss 0 1 2 Patricia M. Roblin 0 1 2 Charlotte A. Gaydos 0 1 2 Peter Cummings 0 1 2 Dorothy L. Patton 0 1 2 Nancy Schulhoff 0 1 2 Jacob Shani 0 1 2 Robert Frankel 0 1 2 Keith Penney 0 1 2 3 Thomas C. Quinn 0 1 2 Margaret R. Hammerschlag 0 1 2 Julius Schachter 0 1 2 0 The Journal of Infectious Diseases 1996; 173:957-62 1996 by The University of Chicago. All rights reserved. 0022-1899/96/7304-0024$01.00 1 Received 25 July 1995; revised 21 November 1995. Presented in part: 33rd Interscience Conference on Antimicrobial Agents and Chemotherapy , New Orleans, October 1993 (abstract 1596); 8th International Symposium on Human Chlamydial Infection, Gouvieux-Chantilly, France, June 1994 (poster 45). Informed consent was obtained from all study participants before enrollment. This study was approved by the Institutional Review Board, State University ofNew York Health Sciences Center at Brooklyn, and the Research Committee, Maimonides Medical Center. 56, Brooklyn, NY 11203-2098 2 Infectious Diseases Division, Departments of Medicine and Pediatrics, State University of New York Health Sciences Center at Brooklyn, and Division of Cardiology, Maimonides Medical Center, Brooklyn; Division of Infectious Diseases, Department of Medicine, Johns Hopkins University School of Medicine , Baltimore , and Laboratory of Immunoregulation, National Institute ofAllergy and Infectious Diseases , Bethesda , Maryland; Department of Obstetrics and Gynecology, University of Washington, Seattle; Department of Laboratory Medicine, University of California , San Francisco 3 Present affiliation: Department of Orthopedic Surgery, New York Medical College , Valhalla To further investigate a proposed relationship between Chlamydia pneumoniae and coronary heart disease, coronary atheromas were collected from patients undergoing percutaneous atherectomy. Fifty-eight atheroma specimens were examined by culture and polymerase chain reaction (PCR) and 22 by electron microscopy. All were negative for C. pneumoniae, except a single specimen that was PCR-positive. These results differ from studies in other populations, in which this organism was identified by nonculture methods within coronary atheromas obtained at autopsy. Anti-C. pneumoniae antibody titers from 65 of the patients were compared with those of 28 asymptomatic controls. IgG titers were higher in controls than in patients. There is no evidence that C. pneumoniae exists within atheromas in this study population, nor does seroprevalence correlate with the presence of coronary disease in these patients. - Chlamydia pneumoniae, a common cause of both upper and lower respiratory tract infection [I, 2], has been investigated as a possible cause of coronary heart disease (CHD) and athero sclerosis. Collaborating investigators have demonstrated a sta tistical correlation between serologic evidence of C. pneumo niae infection and the presence of atherosclerotic vascular disease in populations in Finland and Seattle [3 - 6]. Subsequent reports have described the detection of this organism by several nonculture methods in coronary atheromas obtained from au topsy [7, 8] or atherectomy specimens [9]. These methods included polymerase chain reaction (PCR), immunohistochem istry, and transmission electron microscopy [7 -9]. It has been hypothesized that C. pneumoniae is transported from the lungs into the circulation by pulmonary macrophages, where it then infects vascular tissue [4]. This infection, through mechanisms Methods Patients. Atherectomy specimens were obtained from patients undergoing clinically indicated coronary atherectomy in the Car diac Catheterization Laboratory of the Maimonides Medical Cen ter. Only patients undergoing primary atherectomy on native ves sels were enrolled. On completion of the procedure, atherectomy tissue (-., 10-20 mg) was placed either into Chlamydia transport medium and frozen at -70C or into Kamovsky's glutaraldehyde paraforrnaldehyde fixative and refrigerated. Cultures. Specimens were periodically removed from the freezer and hand-carried the 4 km to our laboratory in an insulated container filled with dry ice. On arrival in the laboratory, the specimens were again stored at -70C until processing. Specimens were then thawed and divided under sterile conditions in a labora tory separate from the Chlamydia laboratory. One portion was placed into PCR buffer and refrozen. The other portion was ho mogenized in a ground glass tissue grinder and then sonicated. Monolayers of cycloheximide-treated HEp-2 cells grown on multiwell microtiter plates were then inoculated and incubated as described previously [10]. Each plate included a positive and a negative laboratory control, and two passes were done on each sample. The monolayers were stained with a fluorescein-conju gated genus-specific anti-Chlamydia antibody for detection of in clusions. PCR. Frozen specimens in PCR buffer were shipped from Brooklyn to Baltimore on dry ice. After thawing, they were ground using disposable grinders. Gloves were changed between each specimen. As previously described, the specimens were treated with 300 j.lL of lysis buffer, consisting of PCR buffer containing proteinase K and NP-40/Tween 40 at concentrations of 100 jjgl mL and 0.5%, respectively, at 60C for I h [11-13]. After heating for 5 min at 100C, 50 jjL was subjected to PCR for a 463-bp sequence of the 16S rRNA gene of C. pneumoniae. Detection of PCR products was by hybridization with a 270-bp nested, biotin labeled RNA probe and subsequent EIA, which used anti-biotin coated microtiter plates and an anti-DNA-RNA monoclonal anti body conjugated to alkaline phosphatase. After addition of a sub strate of methylumbelliferyl phosphate, the end product, methyl umbelliferone, was detected in a fluorometer. Positive values were calculated at 5 SD above the mean of negative controls. Positive laboratory controls were included in each batch in serial dilutions ranging from 200 to 0.1 IFU/reaction. Negative laboratory controls were processed along with patient specimens. PCR was done, using aerosol barrier pipette tips, in a room separate from the laboratory where the EIA was done. Rigorous attention was paid to decontam ination procedures with 1 N HCI between runs [11-13]. Transmission electron microscopy. Atherectomy specimens in Kamovsky's fixative were processed and viewed as described by Kuo et al. [8]. Controls. Patients undergoing preadmission screening for elective surgical procedures or visiting the Family Practice Clinic were recruited as controls. Those aged ~40 with neither a history nor symptoms of CHD or atherosclerosis were enrolled. Nasopharyngeal cultures and serology. Nasopharyngeal spec imens for C. pneumoniae culture were obtained from patients and controls using Dacron-tipped, aluminum-shafted swabs. Cultures were done on HEp-2 cells as described [10]. Blood was collected from patients and controls for C. pneumoniae serology. Serum was assayed for anti-TWl83 Ig (...truncated)