Chlamydia pneumoniae in PBMC: reproducibility of the OMPA nested touchdown PCR.
The aim of our study was to evaluate whether the replicate PCR testing may provide more accurate estimates of C. pneumoniae DNA prevalence in PBMC of patients undergoing carotid endarterectomy. Clinical sensitivity and reproducibility of ompA nested touchdown PCR was also performed. Clinical sensitivity and reproducibility was examined by testing C. pneumoniae-negative PBMC spiked with serial dilutions of semipurified C. pneumoniae elementary bodies (from 8 to 0.002 IFU/ml). Detection of C. pneumoniae DNA was performed by ompA nested 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. pneumoniae DNA performed in replicates of 10 were analysed by probit analysis. C. pneumoniae DNA 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 ompA nested PCR touchdown was 0.008 IFU/ml when 10 replicates were tested. The ompA nested 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 ompA nested touchdown PCR. In conclusion the replicate PCR testing improves the performance of ompA nested touchdown PCR and provides a more accurate estimates of the prevalence of C. pneumoniae in PBMC of patients with atherosclerotic cardiovascular disease.
Sessa
R
R
Department of Public Health Sciences, "La Sapienza" University, Rome, Italy. rosa.sessa@uniroma1.it
Schiavoni
G
G
Di Pietro
M
M
Petrucca
A
A
Cipriani
P
P
Puopolo
M
M
Zagaglia
C
C
Fallucca
S
S
Del Piano
M
M
D001425:Bacterial Outer Membrane Proteins / Q000235:genetics*; D002460:Cell Line; D002690:Chlamydia Infections / Q000453:epidemiology / Q000382:microbiology; D016993:Chlamydophila pneumoniae / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics*; D016894:Endarterectomy, Carotid; D006801:Humans; D009000:Monocytes / Q000502:physiology*; D012044:Regression Analysis; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016036:Seroepidemiologic Studies
D001425:Bacterial Outer Membrane Proteins; D004269:DNA, Bacterial; C078167:OMPA outer membrane proteins