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

Abstract

Inter-and intralaboratory inconsistencies in detection rates of Chlamydia pneumoniae in vascular specimens have been demonstrated. In this study, 66 vascular tissue specimens from 66 patients with vascular disease were tested by three PCR assays: a 16S PCR-based reverse line blot (RLB) assay, a single-step PCR, and a nested PCR. Also, we explored the impacts of different DNA polymerase enzymes on the results based on gel electrophoresis and hybridization. The PCR results by gel electrophoresis in the single-step PCR depended on which DNA polymerase was used. All samples were negative with AmpliTaq Gold DNA polymerase, and 54.5% (36 of 66) were positive with the conventional Taq DNA polymerase. All samples were negative after hybridization with a C. pneumoniae-specific probe. In the nested PCR, all specimens were negative by gel electrophoresis and after hybridization. The RLB assay failed to detect C. pneumoniae in any specimen; however, 20 specimens were Chlamydia sp. positive. The sequence analysis of six of these samples demonstrated Chlamydialike organisms. RLB detected Chlamydia sp. DNA in water and in the elution buffer after passage of the Qiagen columns (11 of 40). This study identified factors that may influence the detection of C. pneumoniae DNA in vascular tissues and consequently bias the perception of a link between C. pneumoniae and vascular diseases. The following are strongly recommended: to use DNA polymerases that have to be activated, to decontaminate with dUTP-uracil-DNA glycosylase, to hybridize with specific probes, to include sufficient controls, and to use molecular grade water.

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

“…In a smaller, but similar, study SEEMUNGAL et al [39] found no relationship between C. pneumoniae DNA detection in the airway at exacerbation and exacerbation frequency. The reported discrepancy in PCR positivity on respiratory samples between the present study and that of BLASI et al [38] may be due to a number of reasons, including differences in PCR techniques used (real time instead of conventional, differences in DNA polymerases, decontamination with 2'-deoxyuridine-5'-triphosphate-uracil-DNA glycosylase, the use of specific probes, inclusion of sufficient controls and the use of molecular grade water) [35,36] and differences in study design and study population. 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.…”

“…Although initial reports were positive, more recent reports, often prospectively designed and adjusted for known cardiovascular risk factors, showed a negative or weak positive association overall between seropositivity for C. pneumoniae and atherosclerosis. Methodology has a strong impact on the possible association between C. pneumoniae and atherosclerosis: detection of the link between C. pneumoniae and coronary artery disease depends on the serological methodology chosen [35][36][37].…”

Involvement of Chlamydia pneumoniae in Atherosclerosis: More Evidence for Lack of Evidence

“…In those studies, unspecific signals may be incorrectly interpreted as C. pneumoniae positive. This is particularly the case when regular Taq DNA polymerase rather than Amplitac Gold DNA polymerase is used (43). In 11 out of 33 studies reviewed by Maraha et al (43), the detection rate was 31.6% when gel electrophoresis only was used to visualize the amplicons.…”

“…This is particularly the case when regular Taq DNA polymerase rather than Amplitac Gold DNA polymerase is used (43). In 11 out of 33 studies reviewed by Maraha et al (43), the detection rate was 31.6% when gel electrophoresis only was used to visualize the amplicons. In the 22 studies in which results were confirmed by hybridization, the detection rate was 24.5% (P ϭ 0.0009).…”

“…In the 22 studies in which results were confirmed by hybridization, the detection rate was 24.5% (P ϭ 0.0009). Furthermore, contamination by environmental chlamydia-like organisms has been detected (43). In a great majority of studies, C. pneumoniae or its DNA was included as a positive control in each amplification run or was added to PCR-negative specimens in a repeat reaction for the detection of PCR inhibitors.…”