Boman et al, working in Sweden, first demonstrated the detection of C. pneumoniae DNA in PBMC of cardiac patients 22. They found a prevalence of 59% of patients undergoing coronary angiography, using a nested PCR targeting an outer membrane protein (ompI) gene. Prevalence was also high in middle-aged blood donors (46%), with a higher prevalence in cases than controls.

A number of investigators have corroborated Boman et al's observation of circulating C. pneumoniae DNA in peripheral blood of cardiac patients, albeit with widely varying estimates of prevalence (Table 1). We identified eight other studies, including seven published manuscripts 21232425262728. and one abstract (Da Costa et al, Proceedings of the Fourth Meeting of the European Society for Chlamydia Research, Helsinki, Finland, 2000), in which cardiovascular patients were compared with controls. These included patients undergoing coronary angiography or angioplasty, vascular patients, patients with acute myocardial infarction or unstable angina, and patients with carotid atherosclerosis at ultrasonography. Controls consisted of blood donors, medical students, apparently healthy controls, or patients with normal coronary angiograms.

The largest of these nine studies examined 1,205 British patients undergoing coronary angiography, and was conducted by Wong et al 28. They found a prevalence of 8.7% in patients with angiographically-proven coronary artery disease, versus 7.2% in those with normal coronary arteries. In sub-group analysis, they found an association between coronary atherosclerosis and C. pneumoniae DNA detection in men but not in women. However, the prevalence of smoking was higher in male cases (78%) than in controls (67%), whereas prevalence was similar in female cases (53%) and controls (51%).

We examined the nine studies by meta-analysis (Figure 1), and found a pooled prevalence of 252 of 1763 (14.3%) in cardiovascular disease patients versus 74 of 874 (8.5%) in controls. The results were not statistically heterogeneous (Breslow-Day X² = 12.3 on 8 degrees of freedom, P= 0.14). The pooled odds ratio, using a random effects model, was 2.03 (95% CI: 1.34, 3.08, P < 0.001), as illustrated in Figure 1. Thus, without adjustment for other cardiovascular risk factors, cardiovascular patients had a higher over-all prevalence of circulating C. pneumoniae DNA than controls subjects.

In Table 2, we summarize nine other identified studies which examined the prevalence of C. pneumoniae DNA in PBMC. In three studies of cardiovascular disease 293031, prevalences of 27.3% to 47.0% were observed. In two studies of chronic obstructive pulmonary disease, prevalences of 24.0% 32 and 47.6% (Blasi et al, Proceedings of the Fourth Meeting of the European Society for Chlamydia Research, Helsinki, Finland, 2000) were observed. The highest prevalence, 60.0%, was found in a small study of patients undergoing peritoneal dialysis 33. Among blood donors, prevalence was 16.7% in Australia 34, 46.2% in Italy 35, and 8.9% in the United States 36. The studies by Bodetti and Timms 34, and by Haranaga et al 36, both corroborated PCR results by detection of C. pnuemoniae-specific antigen in circulating mononuclear cells.

We identified two studies in which DNA was sought concurrently in atheroma and in PBMC 2130. Blasi et al examined 41 patients undergoing aortic surgery 30. Sixteen patients had C. pneumoniae DNA detected concurrently in the resected aneurysmal tissue and in PBMC, three in PBMC alone, and one in arterial wall alone. Thus, 16 of 17 (94.0%) of patients with C. pneumoniae in arterial wall were identified by PCR of PMBC.

Berger et al examined PBMC and resected arterial tissue from 60 vascular disease patients, including carotid and peripheral arteries and aortic aneurysms 21. C. pneumoniae DNA was detected in 12 plaques. Five of these 12 (42.0%) were also PBMC DNA positive. Conversely, 5 of 12 (42.0%) PBMC DNA positive patients were also DNA positive in plaques. No statistically significant correlation between C. pneumoniae DNA presence in plaque or PBMC was found. Furthermore, there was also no correlation between the amount of C. pneumoniae DNA in plaques and in PBMC.

A putative relationship between age and PBMC DNA prevalence was found by Bodetti and Timms 34, with highest prevalence in patients < 35 years (5 of 22, 23%) and > 50 years (3 of 13, 23%) compared with those aged 35 to 50 (2 of 25, 8%), but the sample sizes are too small to allow meaningful inference. Berger et al 21 found that younger patients (mean 59 versus 68 years, P < 0.05) were more likely to have C. pneumoniae DNA in PBMC. By contrast, Iliescu et al 33 found an association between older age and C. pneumoniae DNA detection (62.5 versus 51.9 years, P = 0.01), whereas Haranaga et al 36 found a lower prevalence among older blood donors. No clear relationship with gender was identified in any study.

A relationship between smoking and C. pneumoniae IgG or IgA seropositivity or titre level has been previously demonstrated 3738. Boman et al found an increased prevalence of smoking among patients with C. pneumoniae DNA in PBMC, although the association was not statistically significant 22. Smieja et al found a strong and statistically-significant relationship between C. pneumoniae DNA in PBMC and smoking, with 10.0% prevalence in non-smokers, 9.0% in former smokers, and 25.0% prevalence in current smokers 27. In that study, current smoking was associated with C. pneumoniae DNA positivity independent of age, gender, and season (OR = 4.5, P = 0.004). This relationship was recently confirmed in COPD patients, in whom current smoking was associated with C. pneumoniae DNA detection in PBMC or sputum (OR = 2.6, P = 0.04) 32. Of interest, Berger et al found a relationship between current smoking and the presence of chlamydial DNA in atherosclerotic plaque 21.

Smieja et al found a strong relationship between C. pneumonia DNA detection and season. Among cardiology, respirology and family practice patients in studies spanning four separate Canadian winter seasons 2732 (Smieja et al, unpublished), seasonal variation in prevalence was seen with highest prevalence between February and April (OR = 3.6 to 6.2), and lowest prevalence between June and October. No other reported study has adequately examined seasonal prevalence, but potential inter-month differences were noted in three studies. Rassu et al 35 reported higher prevalence among Italian blood donors in February (57.6%) compared with October (37.9%). Haranaga et al 36 found a lower prevalence in October and November (0 of 137, 0.0%) compared with August or September (21 of 100, 21.0%). Berger et al reported that aneurysmal plaque was DNA positive more often in May (25.0%) or June (50.0%) than in July to October (0.0 to 8.0%, P < 0.05).

Blood sampling, extraction, and PCR targets are summarized in Tables 1 and 2. Generally, Ficoll-hypaque centrifugation of 5 to 10 mL of EDTA blood was used to isolate PBMC. In four studies by two research groups, cell preparation tubes (CPT, BD Vacutainer Systems, Franklin Lakes NJ) were used. No studies were found for a direct comparison between Ficoll-hypaque and CPT methods.

Study investigators sampled 200–1000 μL of PBMC, extracted into a volume of 40–200 μL, and used 3–50 μL for the PCR reaction. Thus, a total volume of 0.15 to 2.50 mL of whole blood was sampled in the final PCR(s) of these various studies. Among eight studies in which sufficient details were given to calculate the total blood sampled, higher DNA prevalence was associated with greater sampled blood volume (X² for linear trend = 55.0, P < 0.001).

Two methods were used for DNA extraction from PBMC: phenol-chloroform or Qiagen QIAamp DNA mini-kits. Examining only cardiovascular patients to enable valid comparison, prevalence was 188 of 1195 (15.7%) extracted with phenol-chloroform, compared with 64 of 568 (11.3%) extracted with Qiagen columns (mean difference = 4.5%, 95% CI: 1.1 to 7.8%).

Eight of 18 studies used a nested PCR based on the ompI gene of the major outer membrane protein (MOMP) described by Tong and Sillis 39, whereas other studies used a variety of nested and non-nested PCRs targeting MOMP, 16s DNA, heat shock proteins, or other targets. Examining only cardiovascular patients, prevalence was higher using the Tong and Sillis nested MOMP PCR (95 of 316, or 30.1%) than with the remaining assays (143 of 1214, or 11.8%), with mean difference = 18.3%, 95% CI: 12.9, 23.7). However, the latter group was heavily influenced by the low prevalence of 79 of 913 (8.7%) in one study 28, which used a nested assay directed at a different MOMP target.

Two studies examined whether C. pneumoniae DNA detection in PBMC correlated with its presence in atherosclerotic plaque, and reached different conclusions. Further studies are required to validate circulating DNA detection as a surrogate for bacterial presence within atherosclerotic plaque.

The prevalence of C. pneumoniae DNA detection in PBMC was found to vary widely: between 0.0% and 46.0% among controls, and between 4.2% and 59.4% in patients with cardiovascular disease. Using meta-analysis, we demonstrated that circulating DNA prevalence was higher among cardiovascular patients than among controls. However, lack of adjustment for potentially confounding variables such as smoking, season, age and gender may negate these results. Indeed, in a recent study of 310 coronary angiography patients and 102 concurrently-recruited family practice controls, adjusting for smoking and season, we found that C. pneumoniae DNA prevalence was less common among heart disease patients than in controls (Smieja et al, unpublished). Addition of this unpublished study would result in a non-significant association between C. pneumoniae detection and cardiovascular disease (pooled OR = 1.6, 95% CI: 0.7, 3.5, P = 0.22). Further studies with concurrent controls, matched or adjusted for age, gender, smoking and season, are required to conclude whether C. pneumoniae DNA detection is associated with cardiovascular disease.

We caution that meta-analysis of non-randomized data may be quite misleading due to heterogeneity of the control groups. Possible explanations for the found association include selection bias, publication bias, confounding by other cardiovascular risk factors, or a true association (whether causal or innocent) between C. pneumoniae and atherosclerosis. Selection bias may be acting in the selection of cases (which represent primarily patients with prevalent, chronic disease and not incident cases), or in the selection of controls. The use of blood donors as controls selects a group in whom many cardiovascular risk factors are less prevalent than in the general population. A second possible bias is of publication bias, in that positive results may have been more likely to get published. This is suggested by examination of a funnel plot (Figure 2), in which the estimated odds ratio of the association between C. pneumoniae DNA detection and vascular disease is graphed against the precision of the estimate. Ordinarily, a funnel shaped with the apex in the middle of the funnel should be seen, as indicated on the diagram 40. The lack of symmetry in the graph suggests that there may be a number of unpublished studies in existence, or that bias in the selection of cases or controls may be playing a role.

Confounding may be playing an important role in any found association with cardiovascular disease. We demonstrated a strong association between PBMC C. pneumoniae DNA detection and current smoking status 27, and Berger et al 21 demonstrated that current smokers are more likely to have C. pneumoniae DNA in atherosclerotic plaque. A more detailed smoking history, or laboratory measures of smoking metabolites such as plasma cotinine, may be useful to better control for confounding by smoking status.

We also found a strong association with Canadian winter or spring season in three of our studies 2732 (Smieja et al, unpublished), with highest prevalence between February and April. Studies by Rassu et al 35 and Berger et al 21 suggest similar seasonal patterns in Italy and Germany, respectively. Seasonal differences may be due to seasonal occurrence of acute C. pneumoniae infections in the community, reactivation of chronic infection, or, potentially, to seasonal differences in DNA extraction efficiency or PCR assay performance. Most studies did not recruit for sufficiently long to observe seasonal patterns, and studies from various geographic locations are required to determine whether seasonality is a wide-spread phenomenon.

Of interest, Boman et al's original study 22 recruited patients between April and May 1998. Similarly, other high prevalence studies (≥ 40.0%) such as Iliescu et al 33 (April to May, 1999), and Blasi et al 30 (December 1998 to February 1999) recruited during times these "high prevalence" months. Conversely, Wong et al 28 recruited between August 1998 and March 1999, and found prevalence of only 8.7%.

Important implications of this seasonal hypothesis are that prevalence will be lowest between June and October, and that controls for patients need to be carefully recruited with matching by month. Berger et al's observation of a peak in C. pneumoniae DNA in atheroma during June 21, and of a lack of correlation with concurrent PBMC DNA, suggests that there may be some delay between PBMC DNA detection and its presence in atheroma. However, Berger et al did not sample between January and April, months during which C. pneumoniae DNA prevalence may have been higher in both PBMC and in atheroma, and during which time a better correlation might have been found between the two sites.

In summary, these data suggest that C. pneumoniae DNA detection in PBMC should be performed, as a minimum, during winter and spring months, and controlled for smoking status. Any comparisons with controls should also be controlled for age and gender, although the relationship between these factors and C. pneumoniae DNA prevalence remains unclear.

Boman and colleagues have reviewed many of the technical aspects of C. pneumoniae DNA amplification in general 841, and of PBMC DNA detection in specific 42. In this review, we examined four important differences in technique which require further investigation: the volume of blood sampled, type of blood specimen used, DNA extraction methods, and PCR primers.

We found that the total volume of blood sampled in the PCR reaction was related to higher detection. We previously demonstrated a relationship between low copy numbers and the proportion of PCR replicates that were positive 43. Solutions to this sampling problem include extracting a large volume of blood, and doing replicate PCRs to maximize detection. However, with larger volumes, inhibitors will also be more concentrated. This may be overcome by testing different concentrations.

Commercial products are now available to simplify and standardize three aspects of testing: BD Vacutainer CPT tubes for blood collection as an alternative to Ficoll-hypaque gradient centrifugation, Qiagen QIAamp DNA mini-kits as an alternative to phenol-chloroform extraction, and a new commercial PCR assay from Abbott Laboratories. The commercial products save labour time but are more expensive. Our review suggests that phenol-chloroform extraction may have a higher yield than Qiagen columns, but inter-study comparisons may be misleading and a direct comparison is needed to validate this finding with paired samples. Experience with a commercial PCR assay has been published, and may enable meaningful comparisons between centres in the future 44.

A MOMP nested PCR described by Tong and Sillis 39 was used in half of the identified studies. Somewhat higher prevalence was seen with this PCR than with other primers, but no firm conclusions should be based on a comparison between studies that used heterogeneous populations. However, they are in accord with our parallel comparison of five in-house published PCRs, in which we found that the Tong and Sillis nested PCR had superior clinical sensitivity for C. pneumoniae in PBMC despite similar analytical sensitivity 45.

Regardless of the best "screening" test for detecting C. pneumoniae DNA in PBMC, additional methods of confirming the identification of C. pneumoniae are desirable 42. Such confirmation would ensure the early identification of false-positive tests, and ensure valid comparisons between tests. However, simply confirming that circulating C. pneumoniae nucleic acids are present does not imply that their detection is clinically relevant. Rather, confirmation is required to define an appropriate molecular measure for prospective clinical studies.

There is good evidence that C. pneumoniae circulates in the bloodstream as a cell-associated infection. We 27 and others 34 have demonstrated that the plasma fractions of PBMC-positive patients were uniformly negative for C. pneumoniae DNA. However, it remains unclear which cells within the peripheral blood mononuclear cell layer contain C. pneumoniae. Peripheral blood mononuclear cells contain monocytes, dendritic cells, and lymphocytes, and may be contaminated with polymorphonuclear cells or platelets. Using CD14 monoclonal selection, Maass et al 29 demonstrated a prevalence of 27.0% C. pneumoniae DNA positivity among patients presenting to hospital with acute coronary syndromes. It is unknown whether this prevalence was higher than in whole blood or buffy coat alone. Kaul et al 24 used adherence to select CD14 cells and found that the CD3+ lymphocytes fraction was positive more often (10 of 28 patients, 35.7%) than the "adherent" fraction of CD14+ cells (3 of 28, 10.7%). Further work is required to determine which cells are infected, and whether cell enrichment would improve detection compared with mononuclear cell preparations.

Potential methods for confirming whether C. pneumoniae is present in PBMC include use of a second DNA-based amplification test, use of RNA-based methods, or detection of antigen. These will be discussed in the same order.

As more sensitive methods are developed, an alternate DNA-based nucleic acid amplification test with a different amplification target may be used to confirm a specimen as a true positive. However, due to low copy number and sampling error, confirmation of only 30–80% of such samples should be expected 1443. Use of higher blood volumes and extraction of a greater proportion of infected cells may improve initial detection as well as confirmation.

Development of methods of quantitating C. pneumoniae in whole blood and PBMC have been developed using TaqMan or LightCycler real-time PCR detection technologies, and Berger et al used a quantitative assay for the detection of C. pneumoniae DNA in PBMC and atherosclerotic plaque 21. Three replicates were run per specimen, and the number of negative runs was not reported. In our experience, only one of three replicates are positive in the majority of patients, indicating very low copy number 2743. For such patients, quantitative methods are unlikely to improve detection, although studies of quantitation versus clinical importance may be informative.

Alternatively, RNA targets may be used for confirmation of C. pneumoniae PBMC positivity. Gieffers et al 46 reported the use of a reverse transcriptase PCR (RT-PCR) targeting 16S RNA transcript, and demonstrated active RNA production by infected PBMC. In volunteers who took antibiotics prior to blood sampling, RNA was still detected, indicating that viable chlamydia were present and that antibiotics had not suppressed chlamydia in infected PBMC. Conversely, as with DNA-based validation, RNA-based confirmation is likely subject to the same sampling problems.

An attractive alternative would be to use a different technology for detection of C. pneumoniae in PBMC. Bodetti and Timms 34 described the detection of circulating C. pneumoniae antigen in PBMC using a specific monoclonal antibody. Ten of 10 PCR positive PBMC specimens were also antigen positive, compared with zero of ten PCR negative specimens. Antigenemia was also assessed and reported in a recent paper by Haranaga et al 36. Wider experience with the sensitivity and specificity of this technique is required, and while it may emerge as a confirmatory test, the method is technically difficult and not suitable for a screening test.