title: 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 ompA-based 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.
authors.affiliation: Department of Hygiene and Medical Microbiology, University of Vienna, Vienna, Austria. petra.apfalter@akh-wien.ac.at
authors.forename: Petra
authors.initials: P
authors.lastname: Apfalter
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Barousch
authors.forename: Marion
authors.initials: M
authors.lastname: Nehr
authors.forename: Athanasios
authors.initials: A
authors.lastname: Makristathis
authors.forename: Birgit
authors.initials: B
authors.lastname: Willinger
authors.forename: Manfred
authors.initials: M
authors.lastname: Rotter
authors.forename: Alexander M
authors.initials: AM
authors.lastname: Hirschl
mesh_terms: D000328:Adult; D000368:Aged; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics*; D016993:Chlamydophila pneumoniae / Q000235:genetics / Q000302:isolation & purification*; D003199:Computer Systems; D004269:DNA, Bacterial / Q000032:analysis; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D016133:Polymerase Chain Reaction / Q000379:methods*; D011933:Reagent Kits, Diagnostic; D012137:Respiratory System / Q000382:microbiology*; D012680:Sensitivity and Specificity; D013183:Sputum / Q000382:microbiology
chemical_list: D001425:Bacterial Outer Membrane Proteins; D004269:DNA, Bacterial; D011933:Reagent Kits, Diagnostic; C078167:OMPA outer membrane proteins