Comparison of a New Quantitative ompA -Based Real-Time PCR TaqMan Assay for Detection of Chlamydia pneumoniae DNA in Respiratory Specimens with Four Conventional PCR Assays

Abstract

Chlamydia pneumoniae, an important respiratory pathogen, is difficult to culture, and detection rates by conventional PCRs vary considerably. A new quantitative ompA-based real-time PCR assay based on TaqMan technology for detection of C. pneumoniae in respiratory samples is described, and its performance in terms of sensitivity and reproducibility is compared with those of four published conventional PCRs (one single-step PCR targeting a cloned PstI fragment; two nested PCRs, one targeting the 16S rRNA gene followed by hybridization and the other targeting the ompA gene; and a touchdown enzyme time-release [TETR] PCR also targeting the 16S rRNA gene). Both ompA-based PCRs showed the best analytical sensitivity. All five assays could detect even lower target levels from spiked sputum, with the 16S rRNA assays performing better than the ompA-based nested PCR (10 ؊6 inclusion-forming units [IFU] were detected in four of four and two of four replicates by the 16S rRNA TETR PCR and the 16S rRNA nested PCR, respectively). In general, the ompAbased real-time protocol produced the most consistent positive results for all replicates tested down to 10 ؊6 IFU. Eight of 45 patient sputum specimens (18%) were C. pneumoniae DNA positive in at least one of four replicates tested by at least one assay. Without taking into consideration the analytical sensitivity or the reproducibility of the test results, the numbers of C. pneumoniae DNA-positive sputum specimens (n ‫؍‬ 8) were four, three, two, two, and one for the 16S rRNA TETR assay, the PstI-based single-step PCR, the ompA-based real-time PCR, the ompA-based nested touchdown PCR, and the 16S rRNA-based nested PCR, respectively. However, the overall rate of concordance of positive results was low. Only one cell culture-positive sputum specimen was positive by four of five assays (14 of 16 replicates; mean cycle threshold value, 25; 10 8 particles/ml of sputum). Thirty-seven specimens were C. pneumoniae negative by all five assays for all replicates tested, as were all negative controls (n ‫؍‬ 65 to 100 per testing panel). No PCR inhibitors were detected by real-time PCR or by the 16S rRNA-based nested assay. We confirm that the analytical sensitivity of an assay for the detection of C. pneumoniae does not necessarily predict its ability to detect its target in sputum. A quantitative, fast, and easy-to-handle diagnostic approach such as the ompA-based real-time TaqMan PCR described here might improve the detection of C. pneumoniae in respiratory samples.

Response to Letters Regarding Article, “Bacterial Signatures in Thrombus Aspirates of Patients With Myocardial Infarction”

“…The C pneumoniae-specific OmpA gene-based real-time PCR, the assay on which we relied, is the most sensitive. 3 In our study, we also analyzed 11 samples with 16S broad range rRNA PCR to confirm our findings.…”

“…The range of C pneumoniaepositive patients varies greatly across studies from 0% to 100%. Apfalter et al 3 reported that 89% of all patients studied had C pneumoniae-specific antibodies, but the pathogen was not detected in a single carotid atheroma by real-time PCR and cell culture in carotid endarterectomy samples. So far, none of the studies have reported the presence of C pneumoniae in thrombus aspirates.…”

“…3 The detection rates by PCRs vary considerably, and the analytic sensitivity of an assay for the detection of C pneumoniae does not necessarily predict its ability to detect its target in every sample. The C pneumoniae-specific OmpA gene-based real-time PCR, the assay on which we relied, is the most sensitive.…”

Chlamydia pneumoniae and Lung Cancer: Epidemiologic Evidence

“…The assays reported in the literature are in-house tests, and most have not been validated compared with culture or even other PCR methods (4). There is evidence of major problems with both inter-and intralaboratory reproducibility (6)(7)(8)(9). Much of the variation in the prevalence of C. pneumoniae DNA detected in human atheroma specimens reported in the literature (0-100%) is thought to be due to variation in methods, although true variation by type of specimen or population studied cannot be excluded.…”

Different inflammatory phenotypes in adults and children with acute asthma

“…Real-time PCR is an effective method for detecting C. pneumoniae using induced sputum [11]. The major outer membrane protein (MOMP) gene has previously been shown to be a good genetic marker for C. pneumoniae in other real-time PCR-based studies [12,13].…”