Real-time polymerase chain reaction and laser capture microdissection: an efficient combination tool for Chlamydophila pneumoniae DNA quantification and localization of infection in atherosclerotic lesions.
Chlamydophila pneumoniae has been implicated in atherosclerosis, but the role of this obligate intracellular pathogen in the development of the above pathology is still unclear. In particular, its presence and quantitative distribution within lesional areas has not yet been defined. We studied 18 carotid biopsies obtained from patients undergoing endoartherectomy. By laser microdissection (LCM), two different sites (intra-plaque and plaque-adjacent areas) were taken from each lesion, and the presence and quantity of the pathogen DNA were determined by real-time polymerase chain reaction (Real-time PCR). A total of 8 plaques, exclusively from patients with unstable angina, were positive in real-time PCR. The bacterial DNA was detected in both lesional areas of 3 plaques which contained the highest number of DNA copies (1,900 to 2,200 copy numbers), while C. pneumoniae DNA was detected only in the intra-plaque area of the other 5 positive (500 to 1,600 copy numbers). No C. pneumoniae DNA was found in the other 10 plaques of which 6 were from patients with unstable angina and 4 from stable angina patients. No DNA from Helicobacter pylori or Cytomegalovirus was found in any plaque. This is the first report where both the target lesion and an adjacent reference site were evaluated for the presence of C. pneumoniae DNA by the combination of LCM and Real-time PCR assays. The integration of these two methodologies offer an excellent tool for in situ studies and may help to elucidate the putative role of C. pneumoniae in atherosclerosis.
Department of Infectious, Parasitic and Immuno-mediated Diseases, Istituto Superiore di Sanità, Viale Regina Elena 299, Rome, Italy. alessandra.ciervo@iss.it
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Ciervo
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Mancini
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Sale
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Russo
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Cassone
D000368:Aged; D050197:Atherosclerosis / Q000382:microbiology* / Q000473:pathology; D002339:Carotid Arteries / Q000382:microbiology / Q000473:pathology; D002690:Chlamydia Infections / Q000382:microbiology* / Q000473:pathology; D016993:Chlamydophila pneumoniae / Q000737:chemistry* / Q000235:genetics; D004269:DNA, Bacterial / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D016894:Endarterectomy, Carotid; D005260:Female; D006801:Humans; D007834:Lasers; D008297:Male; D042282:Microdissection / Q000379:methods*; D008875:Middle Aged; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D004269:DNA, Bacterial