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.

FIG. 1.

ClustalW multiple-sequence alignments of the 85-bp PCR product (QM85), primers (forward primer, QMOMP1; reverse primer, QMOMP2) and probe (QMOMPS) of the real-time PCR assay for the detection of the ompA genes of C. pneumoniae (row 1, MOMP [AF131889]; row 2, MOMP [AF131230]; row 3, MOMP [AF131229]; row 4, IOL-207, CHTMOMPEB [M64064]; row 5, J138, ompA [NC_002491]; row 6, CWL-029, ompA [AE001363]; row 7, AR39, MOMP [AE002167]; row 8, CHTMOMPP [M69230]; row 9, koala type I, CHTOMPAAI [M73038]; row 10, CHTMOMPEQ [L04982]), C. suis (row 11), PCLH197, ompA [AJ440241]), C. psittaci (row 12, strain 84-55, omp1 [Y1656]; row 13, 6BC, MOMP [56980]), C. trachomatis (row 14, C/TW3/OT, omp1 [AF352789]), C. trachomatis (row 15, strain D/Ep6, omp1 [X77364]), C. felis (row 16, Fpn/pring, CPFPNMOMP [X61096]), C. muridarum (row 17, SFPD, CHTMOMPZ [L19221]), C. caviae (row 18, ATCC VR813, ompA [AF269282]), C. pecorum (row 19, LW613, ompA [AJ440240]), and C. abortus (row 20, LLG, omp1 [AF272945]) (the designations in brackets are GenBank accession numbers). Dots and empty spaces indicate identities and gaps compared with the target sequence of the C. pneumoniae strain with GenBank accession no. AF131889, respectively.

FIG. 2.

PCR results obtained with a purified C. pneumoniae DNA dilution series. One IFU per PCR mixture corresponds to 35 chlamydial particles/10 μl, which was used as a template in a total PCR volume of 50 μl. •, ompA real-time PCR (this study); ▴, 16S rRNA-based TETR assay (17); ⧫, PstI-based single-step assay (8); ▪, 16S rRNA-based nested PCR (20); ∗, ompA-based nested touchdown (24).

FIG. 3.

PCR results obtained with sputum spiked with C. pneumoniae-infected HEp-2 cells. •, ompA-based real-time PCR (this study); ▴, 16S rRNA-based TETR assay (17); ⧫, PstI-based single-round assay (8); ▪, 16S rRNA-based nested PCR (20); ∗, ompA-based nested touchdown (23).

FIG. 4.

Overall concordance of positive PCR results obtained with sputa from eight patients (patients L5, L7, L17, L25, B10, W32, W35, and W39). At least one of four replicates was positive for C. pneumoniae DNA by at least one assay. , sputum from which C. pneumoniae was isolated by cell culture. •, ompA-based real-time PCR (this study); ▴, 16S rRNA-based TETR assay (17); ⧫, PstI-based single-step assay (8); ▪, 16S rRNA-based nested PCR (20); ∗, ompA-based nested touchdown (24).