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Reliability of nested PCR for detection of Chlamydia pneumoniae DNA in atheromas: results from a multicenter study
Petra Apfalter1, Ojan Assadian, Francesco Blasi
1Division of Clinical Microbiology, Hygiene-Institute, University of Vienna, Vienna, Austria. Petra.Apfalter@akh-wein.ac.at
Insights
Nested PCR for detecting Chlamydia pneumoniae DNA in endarterectomy specimens is unreliable due to contamination. This study suggests results are questionable, likely caused by amplicon carryover during DNA extraction and PCR.
Area of Science:
- Microbiology
- Molecular Biology
- Cardiovascular Science
Background:
- Chlamydia pneumoniae DNA detection in endarterectomy specimens shows variable rates.
- The reliability of nested PCR for this detection is questioned.
Purpose of the Study:
- Investigate discrepancies in Chlamydia pneumoniae DNA detection rates.
- Identify the source of variability in nested PCR results.
Main Methods:
- Multicenter study analyzing DNA from C. pneumoniae dilution series, spiked tissues, and endarterectomy specimens.
- Utilized three standardized DNA extraction methods and a nested touchdown PCR targeting the ompA gene.
- Compared results within and between laboratories after DNA extract exchange.
Main Results:
- Nested PCR accurately detected C. pneumoniae DNA in dilution series and spiked controls.
- Low positivity rates (2%) in initial endarterectomy specimen analysis.
- After DNA exchange, 13/15 atheroma samples tested positive, but overall positivity (5%) was lower than negative controls (8%).
- No positive results confirmed by 16S rRNA PCR and specific probe hybridization.
Conclusions:
- Positive nested PCR results for C. pneumoniae DNA in endarterectomy specimens are questionable.
- Amplicon carryover during DNA extraction and PCR is the likely cause of false positives.
- Nested PCR is not a reliable method for determining C. pneumoniae prevalence in these samples.
Abstract:
The present multicenter study was designed to find explanations for the discrepancies in the reported rates of detection of Chlamydia pneumoniae DNA in endarterectomy specimens. Coded identical sets of (i) a C. pneumoniae DNA dilution series (panel 1; n = 10), (ii) spiked control tissue specimens (panel 2; n = 10 specimens, including 5 negative controls), and (iii) endarterectomy specimens (panel 3; 15 atheromas, 5 negative controls) were analyzed at four laboratories by three standardized DNA extraction methods in each laboratory and a nested touchdown PCR protocol targeting the ompA gene of C. pneumoniae. Panel 1 samples were correctly identified as positive to levels of 0.3 inclusion-forming units (IFU)/PCR mixture (100%) and 0.03 IFU/PCR mixture (50%). All negative controls were correctly reported as negative. Panel 2 samples were identified as C. pneumoniae positive to levels of 0.01 IFU/PCR mixture (100%) and 0.005 IFU/PCR mixture (91%), independent of the DNA extraction method used, and only one false-positive result was reported. For panel 3 samples, 5 of 240 (2%) analyses (in which DNA extractions and PCR were performed at the same laboratory) were positive; the positive specimens were from three endarterectomy specimens and two negative controls. After exchange of DNA extracts between laboratories, 13 of 15 atheroma samples were C. pneumoniae DNA positive in at least 1 of a series of 48 analyses per atheroma sample; however, the overall positivity rate did not exceed 5% (33 of 720 analyses) and therefore was lower than that for the negative controls (8%; 19 of 240 analyses). Not a single positive result could be achieved when all panel 3 extracts (n = 240 analyses) were reamplified by a 16S rRNA PCR followed by hybridization with a C. pneumoniae-specific probe. Statistical analyses demonstrated that positive results did not occur in an independent and random fashion and could most likely be explained by amplicon carryover at the nested PCR level as well as amplicon introduction during DNA extraction, but not by the patterns of distribution of very low target levels or a certain DNA extraction protocol. The results of studies by nested PCR for detection of the prevalence of C. pneumoniae will always be questionable.