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Several studies have postulated a link between infectious agents and atherosclerosis (3, 12). Epidemiological and pathological studies have shown an association between Chlamydia pneumoniae and atherosclerosis (3, 12). C. pneumoniae has been detected in vascular tissues and degenerative heart valves (3, 9). It has been suggested that Mycoplasma pneumoniae might also play a role in the development of atherosclerosis (12). Myocarditis, pericarditis, cerebral stroke, and vasculitis have been previously associated with M. pneumoniae infection (1, 4, 5, 11). M. pneumoniae might be seen as a plausible candidate to play a role in the pathogenesis of atherosclerosis, because it has been related to cardiovascular disease and its ability to induce chronic inflammation (12). Taylor-Robinson and Thomas have postulated that there is a need to investigate the presence of M. pneumoniae in vascular tissues, because there is a similarity in epidemiological behavior and antibiotic susceptibility between M. pneumoniae and C. pneumoniae (12). Furthermore, M. fermentans has been detected in 1 of 19 human arterial specimens that were positive for C. pneumoniae (10), and it has been shown that M. gallisepticum exhibits tropism for arterial tissue in turkeys (2). However, nothing is known about the presence of M. pneumoniae in human atherosclerotic vascular tissues. Therefore, we investigated the presence of M. pneumoniae in atherectomy specimens and in degenerative heart valve (DHV) specimens.

Atherectomy specimens (n = 39) and DHV specimens (n = 64) were obtained from patients undergoing cardiac surgery for coronary artery bypass graft surgery and heart valve replacement, respectively. Specimens were transported in a Tris-EDTA buffer containing 0.5% sodium dodecyl sulfate. DNA extraction was performed using a commercial kit (QiAmp DNA Minikit; Qiagen, Hilden, Germany). Detection of M. pneumoniae was performed by a PCR assay based on the P1 adhesin gene and using the primers Pn1 (5'-GCC ACC CTC GGG GGC AGT CAG-3') and Pn2 (5'-GAG TCG GGA TTC CCC GCG GAG G-3') as described previously (7). PCR products were visualized after electrophoresis in 2% ethidium bromide-stained agarose gels (Fig. 1). To confirm the PCR results, the PCR product was spotted and hybridized with the 5'-biotinylated probe MP2-B (5'-GGT GAA GGA ATG ATA AGG CT-3'). Hybridization signals were visualized using streptavidin-peroxidase and enhanced chemiluminescence (ECL) detection reagents (Amersham, Little Chalfont, United Kingdom).

View larger version (53K): [in this window] [in a new window]   FIG. 1.   Agarose gel electrophoresis of PCR products obtained with P1 adhesin gene primers. (A) Lane M, 1-kb ladder; lanes 1 to 5, vascular tissues of five patients; lane 6, negative control. (B) Lanes 1 to 6 contain the same samples shown in panel A spiked with M. pneumoniae target DNA (positive controls).

M. pneumoniae was detected in only 1 (2.5%) of the 39 atherectomy specimens and in 2 (3%) of the 64 DHV specimens. This is the first study on detection of M. pneumoniae in vascular tissues. Thus, there are no other data available to compare our results with.

In general, several factors can influence the results of PCR-based studies on detection of microorganisms in vascular tissues, including contamination, inhibition, size and composition of specimens, and the lack of standardized methods for DNA extraction and PCR assays (8). We used a validated PCR assay with a high sensitivity (92 to 100%) (7). Strict PCR anticontamination precautions were taken, such as the use of UDG/dUTP, to prevent carryover of previous PCRs. Sample processing and the PCR assay were performed in separate rooms. A negative control was processed with every set of five samples. To control for inhibition of the PCR, a second sample of each specimen was spiked with M. pneumoniae target DNA, and inhibited PCR samples were again purified and retested. Also, this PCR assay was used in our laboratory on respiratory samples. A total of 179 throat samples obtained from patients with respiratory tract infections were tested. These samples were sent to the laboratory for detection of respiratory pathogens. The M. pneumoniae PCR detected M. pneumoniae in 27 (16%) of the 179 samples. These results indicate that the PCR assay we used was sensitive and able to detect M. pneumoniae. However, it should be mentioned that there is no similarity in composition between vascular tissues and throat specimens.

It has been found in a serological study that, in contrast to C. pneumoniae antibodies, M. pneumoniae antibodies are not associated with recurrent events in patients with unstable angina (6). Using PCR, we were unable to detect M. pneumoniae in the great majority of the 103 tested specimens. The results of this study do not support the hypothesis that M. pneumoniae is an important factor in the development of vascular disease. Further investigations are needed to confirm our results.