Rapid Detection of Chlamydia pneumoniae DNA in Peripheral Blood Mononuclear Cells of Coronary Artery Disease Patients by Real-Time Fluorescence PCR
WATTANA LEOWATTANA, M.D.*, SASIKANT POKUM, B.Sc. *, NITHI MAHANONDA, M.D.**, NARAVAT POUNGVARIN,M.D.*
Affiliation : * Division of Clinical Chemistry, Department of Clinical Pathology, **Her Majesty Cardiac Center, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand.
Several recent reports including serological, pathological and animal studies have asso- ciated Chlamydia pneumoniae with coronary artery disease (CAD). In order to establish whether chronic C. pneumoniae infection is linked to coronary artery disease, clinical intervention trials may be needed. However, to detect eligible patients with persistent infection, a reliable diagnostic marker must be developed for identifying cases and assessing efficacy of antichlamydial therapy. Moreover, the prevalence of circulating C. pneumoniae DNA in CAD patients varied widely from previous reports. A real-time PCR has been established by using HL-1 and HR-1 primer to amplify 437 base pairs product. Confirmation of the product was performed on LightCycler by melting curve analysis of detection probes labeled with LC-Red705. Ninety-five angiographically con- firmed CAD patients and 104 normal, healthy volunteers were recruited. The mononuclear cell layer was separated from collected blood and rapid, single step real-time PCR was used to detect C. pneumoniae DNA. C. pneumoniae DNA in peripheral blood mononuclear cells (PBMC) was found in 17 per cent of 95 CAD patients and 1 per cent of 104 normal healthy volunteers (odds ratio 20.86, 95% confidence interval 2.71 - 160.67, p < 0.0001). There was no association between C. pneumoniae DNA in PBMC and serological status. The rapid, real-time PCR showed a clear- cut result between positive and negative cases. PBMC-based real-time PCR may be a useful tool for identifying subjects carrying C. pneumoniae in the circulation or in the vascular wall as well. It will be a specific indicator of current infection and will be used as a marker for assessing the microbiological efficacy of antichlamydial therapy in clinical intervention trials.
Keywords : Chlamydia pneumoniae DNA, Peripheral Blood Mononuclear Cells and Real-Time Fluorescence PCR
