Di Pietro M

Detection of Chlamydia pneumoniae in atherosclerotic coronary arteries.

Chlamydia pneumoniae has recently been associated with the development of coronary heart diseases by sero-epidemiological studies and by direct detection of the organism in atherosclerotic tissues. The aim of our study was to employ a semi-nested PCR approach to investigate the presence of C. pneumoniae in both normal and atherosclerotic coronary arteries of humans obtained at autopsy. Moreover, we have evaluated the role of infection with C. pneumoniae in relation to the extent of coronary atherosclerosis. One hundred and eighty coronary artery specimens were collected at autopsy from 60 consecutive subjects (three arterial segments from each subject). Atherosclerosis in each arterial segment was graded histologically by the Stary classification. Thirty normal coronary arteries were also taken at autopsy as control. PCR results evidenced the presence of C. pneumoniae DNA in atherosclerotic coronary arteries in 19 (31.7%) of 60 subjects examined, while none of the 30 subjects with non-atherosclerotic tissues was positive (p=0.001). Moreover, of the 180 atherosclerotic specimens examined, C. pneumoniae DNA was detected in 3.4% (2/59) of mild atherosclerotic lesions, and in 14.0% (17/121) of advanced atherosclerotic lesions (p=0.05). Our results demonstrate that the presence of C. pneumoniae DNA may be associated with the severity of coronary atherosclerosis.

Chlamydia pneumoniae in asymptomatic carotid atherosclerosis.

We evaluated, in 415 patients with asymptomatic carotid atherosclerosis: (i) the prevalence of C. pneumoniae DNA in atherosclerotic carotid plaques and peripheral blood mononuclear cells (PBMC); (ii) the distribution of C. pneumoniae in atherosclerotic carotid plaques and PBMC from the same patients; (iii) the correlation between circulating anti-chlamydial antibodies and the presence of C. pneumoniae DNA. Overall, 160 atherosclerotic carotid plaques and 174 PBMC specimens from patients with asymptomatic carotid atherosclerosis were examined by ompA nested touchdown PCR for presence of C. pneumoniae. In addition, C. pneumoniae DNA was detected in 81 specimens of atherosclerotic carotid plaque and PBMC obtained from the same patients. C. pneumoniae DNA was found in 36.9% of atherosclerotic carotid plaques and in 40.2% of PBMC specimens examined (P=NS). With regard to 81 patients, C. pneumoniae DNA was detected in 27.2% of atherosclerotic carotid plaques and in 44.4% of PBMC specimens(P=0.05). In 18 patients, the presence of C. pneumoniae DNA in PBMC specimens and atherosclerotic carotid plaques coincided (P=0.005). No statistically significant association was found between anti-C. pneumoniae antibodies (IgG and IgA) and positive PCR results. In conclusion, our results suggest that the detection of C. pneumoniae DNA in PBMC specimens seems to be a first-choice method to identify the patients at risk for endovascular chlamydial infection.

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.

Chlamydia pneumoniae infection in patients with acute coronary syndrome: a clinical and serological 1-year follow-up.

The role of Chlamydia pneumoniae infection in pathogenesis and prognostic stratification of patients with acute coronary syndromes is still unclear. However, a limitation of many studies is the evaluation of the long-term prognostic role of a sample obtained during the acute phase, whereas the assessment of the temporal trend of antibody titers could be more useful. One-hundred and fourteen consecutive patients with acute coronary syndromes (71 with acute myocardial infarction and 43 with unstable angina) were studied. Blood samples were obtained immediately after hospital admission and 1, 3, 6 and 12 months after the acute event. The microimmunofluorescence test was used to detect C. pneumoniae specific antibodies. The incidence of new coronary events (death, myocardial infarction, recurrent angina) was recorded during the 1-year follow-up period. No significant difference was found between patients with (n = 35) or without (n = 79) new coronary events (N.C.E.) regarding baseline and serial values of C. pneumoniae antibodies. The rate of high titers at any time of follow-up was also similar in the two groups: IgG ≥1:512 were present in 52%, 64%, 55% and 32% of N.C.E.+ patients, and in 48%, 54%, 52% and 36% of N.C.E.- patients at 1, 3, 6 and 12 months respectively; IgA ≥ 1:256 were present in 26%, 23%, 30% and 23% of N.C.E.+ patients and in 20%, 30%, 25% and 19% of N.C.E.- patients at 1, 3, 6 and 12 months respectively. Our data indicate that elevated titers of C. pneumoniae antibodies, even with a serial 1-year evaluation, are not a predictor of future coronary events in patients with acute myocardial infarction or unstable angina.