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Detection of Chlamydia pneumoniae in atherosclerotic tissue: a comparative study of PCR and immunocytochemistry
1Division of Cardiac and Vascular Sciences, St George's Hospital, University of London, Cranmer Terrace, UK.
Abstract:
The reported prevalence of Chlamydia pneumoniae in atherosclerotic tissue appears to depend on the detection system used. This introduces problems in determining the role of C. pneumoniae in atherosclerosis. This study analyses the sensitivity and performance of molecular diagnostic methods for the detection of C. pneumoniae and polymerase chain reaction (PCR) inhibitors in atheromatous tissue. Atherosclerotic tissue taken from 30 coronary endarterectomies, nine coronary arteries from explanted hearts, 16 carotid and two femoral endarterectomies are studied. Nested PCR (nPCR) assays targeting the PstI restriction fragment, the OmpA gene and the CRP operon of the chlamydial genome and immunocytochemistry (ICC) are used. Internal controls (IC) are constructed to co-amplify with the specific amplicons and identify the presence of inhibitor. The OmpA, PstI and CRP operon PCR assays had similar analytical sensitivities. However, the OmpA PCR was most affected by PCR inhibitors. Despite this, eight samples (14%) tested positive in the OmpA nPCR and no positives were found using the PstI or CRP operon nPCRs. Primary isolates of C. pneumoniae obtained from 12 patients with acute respiratory infection were positive in all three assays. Of the 48 specimens available for ICC, 33 (69%) were positive for chlamydial antigens. These included samples found positive by PCR. Dilution of samples to eliminate PCR inhibitors may have contributed to the discordant ICC and PCR results. The OmpA PCR, when used with an IC to identify samples with PCR inhibitors, is a reliable tool. However, the sensitivity of the ICC methods justifies their continued use.
Insights
Detecting Chlamydia pneumoniae in atherosclerosis is challenging due to varied methods. The OmpA PCR with internal controls is reliable, but immunocytochemistry shows higher sensitivity for detecting this bacterium in atherosclerotic tissue.
Area of Science:
- Microbiology
- Cardiovascular Pathology
- Molecular Diagnostics
Background:
- The prevalence of Chlamydia pneumoniae in atherosclerotic tissue is inconsistent across studies, complicating its role in atherosclerosis.
- Accurate detection methods are crucial for understanding the pathogenesis of atherosclerosis.
Purpose of the Study:
- To evaluate the sensitivity and performance of molecular diagnostic methods for Chlamydia pneumoniae detection in atheromatous tissue.
- To assess the impact of polymerase chain reaction (PCR) inhibitors on diagnostic accuracy.
Main Methods:
- Analysis of atherosclerotic tissue from various endarterectomies and explanted hearts.
- Application of nested PCR (nPCR) assays targeting PstI, OmpA gene, and CRP operon.
- Utilizing immunocytochemistry (ICC) for chlamydial antigen detection.
- Incorporation of internal controls (IC) to identify PCR inhibitors.
Main Results:
- OmpA, PstI, and CRP operon PCR assays demonstrated similar analytical sensitivities.
- OmpA PCR was most susceptible to PCR inhibitors, yet detected C. pneumoniae in 8% of samples.
- Immunocytochemistry (ICC) detected chlamydial antigens in 69% of specimens, including PCR-positive samples.
- Primary C. pneumoniae isolates were positive across all PCR assays.
Conclusions:
- The OmpA PCR, when employing an internal control to manage PCR inhibitors, proves to be a dependable diagnostic tool.
- Despite PCR challenges, the high sensitivity of ICC methods warrants their continued application in detecting C. pneumoniae in atherosclerotic tissues.
