title: Analytical sensitivity, reproducibility of results, and clinical performance of five PCR assays for detecting Chlamydia pneumoniae DNA in peripheral blood mononuclear cells.
abstract: Chlamydia pneumoniae has been associated with atherosclerosis and coronary artery disease (CAD), and its DNA has been detected in atheromatous lesions of the aorta, carotid, and coronary arteries by a variety of PCR assays. The objective of this study was to compare the performances of five published PCR assays in the detection of C. pneumoniae in peripheral blood mononuclear cells (PBMCs) from patients with coronary artery disease. The assays included two conventional PCRs, one targeting a cloned PstI fragment and one targeting the 16S rRNA gene; two nested PCRs, one targeting the 16S rRNA gene and one targeting ompA; and a touchdown enzyme time release (TETR) PCR, targeting the 16S rRNA gene. All PCRs had similar analytical sensitivities and detected a minimum of 0.005 inclusion-forming units (IFU) of C. pneumoniae; the ompA nested PCR and the TETR PCR were slightly more sensitive and detected 0.001 IFU. Assay reproducibility was examined by testing 10 replicates of C. pneumoniae DNA by each assay. All five assays showed excellent reproducibility at high levels of DNA, with scores of 10 out of 10 for 0.01 IFU, but exhibited decreased reproducibility for smaller numbers of C. pneumoniae IFU for all tests. Pairwise comparison of test results indicated that there was a significant difference between tests (Cochran Q = 32.0, P<0.001), with the PstI fragment (P<0.001) and 16S rRNA (P = 0.002) assays having lower reproducibility than the nested ompA and TETR assays. To further analyze assay sensitivity, C. pneumoniae-infected U-937 mononuclear cells were added to whole blood, and extracted mononuclear-cell DNA was tested by each assay. All five assays showed similar sensitivities, detecting 15 infected cells; three assays detected 3 infected cells, while all assays were negative at the next dilution (1.5 infected cells). A striking difference in performance of the five assays was seen, however, when PBMCs from CAD patients were tested for C. pneumoniae DNA. The ompA nested PCR detected C. pneumoniae DNA in 11 of 148 (7.4%) specimens, the 16S rRNA nested PCR detected 2 positives among the 148 specimens (1.4%) (P<0.001), and the other 3 assays detected no positive specimens (P<0.001, compared with the ompA assay). These results indicate that analytical sensitivity alone does not predict the ability of an assay to detect C. pneumoniae in whole-blood-derived PBMCs. Before standardized assays can be used in wide-scale epidemiological studies, further characterization of these assays will be required to improve our understanding of their performance in the detection of C. pneumoniae in clinical material.
authors.affiliation: Hamilton Regional Laboratory Medicine Program, St. Joseph's Hospital, Hamilton, Ontario, Canada. mahonyj@fhs.mcmaster.ca
authors.forename: J B
authors.initials: JB
authors.lastname: Mahony
authors.forename: S
authors.initials: S
authors.lastname: Chong
authors.forename: B K
authors.initials: BK
authors.lastname: Coombes
authors.forename: M
authors.initials: M
authors.lastname: Smieja
authors.forename: A
authors.initials: A
authors.lastname: Petrich
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000235:genetics; D002690:Chlamydia Infections / Q000150:complications; D016993:Chlamydophila pneumoniae / Q000235:genetics / Q000302:isolation & purification*; D003327:Coronary Disease / Q000382:microbiology*; D004269:DNA, Bacterial / Q000032:analysis*; D020459:Genes, rRNA; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D020298:U937 Cells
chemical_list: D001425:Bacterial Outer Membrane Proteins; D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S; C078167:OMPA outer membrane proteins