Reliability of Nested PCR for Detection of Chlamydia pneumoniae DNA in Atheromas: Results from a Multicenter Study Applying Standardized Protocols

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.During the past decade the association between Chlamydia pneumoniae, an important respiratory pathogen in humans (10, 11), and atherosclerosis (8) has continually been strengthened by direct detection of the organism in atherosclerotic tissues (21). Although there are no standardized PCR protocols, nested PCR has so far been the method most often used for this purpose. However, comparison of the data has been difficult because detection rates have varied considerably by protocol and individual testing center (4, 9, 12, 14-19, 21, 22, 24). Until now no systematic evaluation of various DNA extraction protocols and kits or of PCRs has been conducted for atherosclerotic tissue specimens. It is still not clear if methodological difficulties, a target distribution problem, various C. pneumoni...

Is the Perceived Association between Chlamydia pneumoniae and Vascular Diseases Biased by Methodology?

“…Hybridization is also an important measure to minimize false PCR-positive signals, and the advantages of confirmation of PCR-positive signals by hybridization with a specific probe have been described before (4). Analyzing 33 studies with regard to hybridization in the detection of C. pneumoniae DNA, we found 11 studies in which only gel electrophoresis had been used to visualize PCR products (8,16,18,30,37,38,40,(42)(43)(44)(45).…”

“…The DNA amplification of C. pneumoniae-specific sequences in vascular tissue specimens, however, has been shown to vary greatly among study groups. More recent reports have criticized the lack of standardization of C. pneumoniae PCR methodology (3,4,15).…”

The role of atypical respiratory pathogens in exacerbations of chronic obstructive pulmonary disease

“…In the current authors' view, real-time PCR is the current standard in the clinical microbiology laboratory, and the question of cross-contamination occurs whenever a nested PCR is performed. In this sense, the results of studies that use nested PCR to determine the prevalence of C. pneumoniae will always be questionable [40].…”

Chlamydia pneumoniae and Lung Cancer: Epidemiologic Evidence

“…The assays reported in the literature are in-house tests, and most have not been validated compared with culture or even other PCR methods (4). There is evidence of major problems with both inter-and intralaboratory reproducibility (6)(7)(8)(9). Much of the variation in the prevalence of C. pneumoniae DNA detected in human atheroma specimens reported in the literature (0-100%) is thought to be due to variation in methods, although true variation by type of specimen or population studied cannot be excluded.…”