Analytical sensitivity, reproducibility of results, and clinical performance of five PCR assays for detecting Chlamydia pneumoniae DNA in peripheral blood mononuclear cells

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, T he objective of this study was to compare the performances of five publishe d PCR assays In the detection of C. pneumoniae in peripheral blood mononucl ear cells (PBMCs) from patients with coronary artery disease. The assays in cluded two conventional PCRs, one targeting a cloned PstI fragment and one targeting the 16S rRNA gene; two nested PCRs, one targeting the 16S rRNA ge ne and one targeting ompA; and a touch-down 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. pneumo niae; the ompA nested PCR and the TETR PCR were slightly more sensitive and detected 0.001 IFU, Assay reproducibility was examined by testing 10 repli cates of C. pneumoniae DNA by each assay, All five assays shelved excellent reproducibility at high levels of DNA, with scores of 10 out of 10 for 0.0 1 IFU, but exhibited decreased reproducibility for smaller numbers of C. pn eumoniae IFU for all tests. Pairwise comparison of test results indicated t hat 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 furt her analyze assay sensitivity, C. pneumoniae-infected U-937 mononuclear cel ls 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 we re negative at the next dilution (1.5 infected cells), A striking differenc e 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 dete cted 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 p redict the ability of an assay to detect C. pneumoniae in whole-blood-deriv ed PBMCs, Before standardized assays can be used in wide-scale epidemiologi cal studies, further characterization of these assays will be required to i mprove our understanding of their performance in the detection of C. pneumo niae in clinical material.