Chlamydia Pneumoniaein PBMC: Reproducibility of the OMPANested Touchdown PCR

The aim of our study was to evaluate whether the replicate PCR testing may provide more accurate estimates of C. pneumoniaeDNA prevalence in PBMC of patients undergoing carotid endarterectomy. Clinical sensitivity and reproducibility of ompAnested touchdown PCR was also performed. Clinical sensitivity and reproducibility was examined by testing C. pneumoniae-negative PBMC spiked with serial dilutions of semipurified C. pneumoniaeelementary bodies (from 8 to 0.002 IFU/ml). Detection of C. pneumoniaeDNA was performed by ompAnested touchdown PCR. Each clinical and spiked PBMC DNA specimen was analyzed in replicates of 1,3,5 and 10. PCR results of serial dilutions of C. pneumoniaeDNA performed in replicates of 10 were analysed by probit analysis.C. pneumoniaeDNA was detected in 14 of the 30 (46.7%) PBMC clinical specimens examined when 10 replicates were tested. When we analyzed 1, 3 and 5 replicates, 4 (13.3%), 7(23.3%), 12(40%) of the 30 specimens were positive, respectively. The limit of detection of ompAnested PCR touchdown was 0.008 IFU/ml when 10 replicates were tested. The ompAnested PCR had reproducibility scores of 10 for 10 from 8 to 4 IFU/ml concentration, but scores decreased for smaller numbers of IFU/ml. Our results showed that repeat testing of the same specimen increased clinical sensitivity as well as reproducibility of the ompAnested touchdown PCR. In conclusion the replicate PCR testing improves the performance of ompAnested touchdown PCR and provides a more accurate estimates of the prevalence of C. pneumoniaein PBMC of patients with atherosclerotic cardiovascular disease.