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Detection of the organism in tissue

C. pneumoniae was first detected in vascular tissue in 1992 [Shor et al., 1992] and since then, it has frequently been found in diseased blood vessels. It has been observed that, based on published reports, C. pneumoniae is twenty times more likely to be found in atherosclerotic compared with normal vascular tissue [Danesh et al., 2000]. However, atherosclerosis is ubiquitous and truly normal vascular tissue is difficult to find. This is compounded by the fact that vascular samples are generally not available from live subjects unless obtained during interventions such as coronary atherectomy or bypass surgery and even then, normal tissue is usually not obtainable. In fact, we found that just under half of all published studies reviewed up to September 1998 [Wong, Gallagher & Ward, 1999] did not use control tissue [Weiss et al., 1996; Kuo et al., 1995; Jackson et al., 1997; Blasi et al., 1996; Davidson et al., 1998; Bauriedel et al., 1998; Lindholt et al., 1998 & Paterson et al., 1998] and in only three studies were case and control tissues matched for age, source of tissue and method of detection [Kuo et al., 1995; Maas et al., 1997 & Petersen et al., 1998]. To date, this situation remains unchanged and no subsequent study has had control tissues fulfilling these criteria [Ericson et al., 2000; Ouchi et al., 1998; Linares-Palomino et al., 2001; Radke et al., 2001; Valassina et al., 2001; Vink et al., 2001; Ouchi et al., 2000; Ozsan et al., 2000; Jantos et al., 2000; Farsak et al., 2000; Daus et al., 2000; Chiu, 1999 & Bauriedel et al., 1999]. Nevertheless, it is possible to assess the role of C. pneumoniae by examining its prevalence amongst populations at differing risk for vascular disease. In making such comparisons, it is important to take into account the investigators involved and the methods used. This is because examination of the same tissue by different groups and methods can result in widely varying results [Apfalter et al., 2001]. The Seattle group has examined the prevalence of the organism in coronary arteries from native Alaskans dying mainly from non cardiovascular causes (mean age 34.1 years, range 15 to 57 years) [Davidson et al., 1998], from young Americans (age 15 to 34 years) who also did not die from coronary disease [Kuo et al., 1995] and from Americans who had coronary atherectomy (median age 55, range 35 to 81) [Campbell et al., 1995]. The reported prevalence for these 3 groups using PCR were 23.3%, 0 to 17% and 32% respectively. It can be seen that there appears to be a gradient with respect to age, but the prevalence in the Eskimos, a low risk group for coronary heart disease, was not much lower than that of the older Americans with coronary disease. However, the young Americans study was among those with adequate control tissue and all three such studies did find an association with atherosclerosis.

Another approach for assessing the role of C. pneumoniae in atherosclerosis is to correlate the presence of the whole organism or its products with the extent or severity of coronary artery disease. Here again the results are conflicting. In our laboratory, the presence of the organism was focal and not associated with either the severity or extent of disease after allowing for the differential effects of PCR tissue inhibitors [Thomas et al., 1999]. Similar results were found by others [Davidson et al., 1998; LaBiche et al., 2001] but associations have been reported [Ericson et al., 2000; Vink et al., 2001 & Maas et al., 1997]. In some studies the organism was said to be more prevalent in unstable compared with stable plaques [Radke et al., 2001; Bauriedel et al., 1999]. What is clear from these studies is that in any individual, C. pneumoniae has never been found at all atherosclerotic sites. This suggests that infection occurs after the development of atherosclerosis and if anything, it is an exacerbating rather than a causal factor. However Wong et al., 1999a in a large study were able to demonstrate that circulating C. pneumoniae DNA in peripheral blood monocytes was associated with angiographic abnormalities of the coronary arteries in males referred to an angina clinic. This has been variously confirmed and refuted since.

Despite these numerous studies, the prevalence of C. pneumoniae in blood vessels and therefore, the size of any potential public health problem remains in doubt. In the blinded, multicentre PCR comparison study [Apfalter et al., 2001], fifteen atheroma specimens were tested by nine centres. Three centres using a total of seven different PCR protocols failed to detect the organism at all, while one centre using four protocols found the organism inconsistently in two samples. The remaining centres found C. pneumoniae in one to nine samples. This is clearly not a satisfactory state of affairs. Results were still inconsistent when centres using the same PCR protocol were compared. The reasons for these varying results could not be determined but, as only a few studies prior to this study had failed to find C. pneumoniae in atherosclerotic tissue [Weiss et al., 1996; Daus et al., 1998 & Paterson et al., 1998]  it is possible that there are other negative studies which have not been published due to publication bias. 

Overall, Kalayoglu et al., 2002 point out that data collected from 43 studies published before October 2002 indicate a high prevalence of C. pneumoniae in atheromatous tissue (46% of 1852 specimens) but not in healthy arteries (less than 1% of 239 specimens). They note:

• Huge variability exists in the direct detection methodology used by individual investigators and in the proportion of samples with detected infection. There have rarely been adequate controls for tissue inhibitors

• Detection results correlate very poorly with serology

• C. pneumoniae is present in specimens recovered from both young and old

• C. pneumoniae is found in a wide range of blood vessels, including those  which have a low rate of atherosclerosis

[YW]. Updated [MEW] August 2003

Conclusion

There is little doubt that C. pneumoniae is commonly found in the walls of coronary arteries and other vessels and that it is associated with atherosclerotic lesions. Although striking, this association is  not exclusive since the organism can be found in vessels such as the internal mammary artery which rarely suffer significant atherosclerosis. Much of the observed variability in studies can be attributed to four factors:

• Many studies are poorly controlled or are statistically inadequate

• The amount of C. pneumoniae present is often small and approaching detection limits

• The absence of validated standard protocols for performing PCR or immunocytochemistry for C. pneumoniae. The study of Apfalter et al 2001 indicates the huge variability in the home made PCRs that researchers have generally used for the detection of C. pneumoniae DNA.

• The effect of tissue inhibitors on detection. Severely atherosclerosed vessels, particularly those containing calcium, are likely to be more inhibitory for PCR tests than those with less atheromatous plaque; few studies have controlled for this. 

Even were the detection technology perfect, it is difficult to determine whether an observed association of C. pneumoniae with atherosclerosis is because the organism causes atheroma or, alternatively, that it has a predilection for pre-formed atherosclerotic plaque, perhaps because the latter is an immunologically active site to which macrophages carrying phagocytosed chlamydiae are attracted. Although quantitative PCR studies are being performed to help illuminate the relationship between C. pneumoniae infection and the severity of atherosclerosis [for example Berger et al., 2000 who found no association with severity], they are unlikely to resolve the dilemma of whether the organism causes atheroma or is a relatively harmless passenger.

From the direct detection studies we can be reasonably certain: 1) That C. pneumoniae is present in a high proportion of people with significant atheromatous plaque; 2) That it is associated within the plaque with smooth muscle cells and with macrophage-derived foam cells and 3) That the amounts of C. pneumoniae present in vessels are usually relatively small. Whether this amount is sufficient to significantly exacerbate atherosclerosis or to precipitate plaque rupture is unknown but is being pursued from a variety of approaches.

[MEW] August 2003

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References

Apfalter, P., Blasi, F., Boman, J., Gaydos, C. A,. Kundi, M., Maass, M. et al. (2001). Multicenter comparison trial of DNA extraction methods and PCR assays for detection of Chlamydia pneumoniae in endarterectomy specimens. Journal of Clinical Microbiology 39, 519 - 524. [Shows the huge variability in PCR detection methods for Ch. pneumoniae in different laboratories, presumably underlying many of the apparent contradictions in the literature]. Full article

Bauriedel, G., Schmucking, I., Hutter, R., Schmidt, T. & Welsch, U. (1998). Evidence of Chlamydia pneumoniae in Human Arteriosclerotic Lesions: Insights into Its Possible Pathogenic Role [abstr]. Journal of the American College of Cardiology 31, A390 - A391.

Bauriedel, G., Welsch, U., Likungu, J. A., Welz, A. & Luderitz, B. (1999). Chlamydia pneumoniae in coronary plaques: Increased detection with acute coronary syndrome. Deutsch Medizinische Wochenschrrift 124, 375 - 380. [In German. C. pneumoniae detected in 32/51 (63%) coronary primary lesions of symptomatic patients, particularly in sites that revealed small healing activity and / or propensity to plaque rupture. Suggests a pathogenic role of Ch. pneumoniae in coronary plaque rupture.]

Berger, M., Schroder, B., Daeschlein, G., Schneider, W., Busjahn, A., Buchwalow, I., Luft, F. C. & Haller, H. (2000). Chlamydia pneumoniae DNA in non-coronary atherosclerotic plaques and circulating leukocytes. Journal of Laboratory and Clinical Medicine 136, 194 - 200.

Blasi, F., Denti, F., Erba, M., Cosentini, R., Raccanelli, R., Rinaldi, A. et al. (1996). Detection of Chlamydia pneumoniae but not Helicobacter pylori in atherosclerotic plaques of aortic aneurysms. Journal of Clinical Microbiology 34, 2766 - 2769. Full article [Ch . pneumoniae DNA in 21/56 patients with aortic aneurysms but no H. pylori DNA . No control group]

Campbell,  L. A., O'Brien, E. R., Cappuccio, A. L., Kuo, C. C., Wang, S. P., Stewart, D. et al. (1995). Detection of Chlamydia pneumoniae TWAR in human coronary atherectomy tissues. Journal of Infectious Diseases 172, 585 - 588.

Chiu, B. (1999). Multiple infections in carotid atherosclerotic plaques. American Heart Journal 138, (Supplement) S534 - S536. [Multiple infectious agents can be found in atheromatous plaque including the dental pathogens P. gingivalis and S. sanguis. Demonstrates the fallacy of focusing on just the chlamydiae in coronary artery disease].

Danesh, J., Whincup, P., Walker, M., Lennon, L., Thomson, A., Appleby, P.,  Wong, Y., Bernades-Silva, M. & Ward, M. E. (2000). Chlamydia pneumoniae IgG titres and coronary heart disease: prospective study and meta-analysis. British Medical Journal 2000;321:208-13.   Full article [Plus associated journal editorial and commentary on and offline. One of the largest prospective studies, general practice based, plus meta-analysis of previous data including Wald et al., 2000. Concludes that contribution of C. pneumoniae to coronary heart disease must be small if anything.]

Daus, H., Ozbek, C., Saage, D., Scheller, B., Schieffer, H., Pfreundschuh, M.  et al. (1998). Lack of evidence for a pathogenic role of Chlamydia pneumoniae and cytomegalovirus infection in coronary atheroma formation. Cardiology 90, 83 - 88.[No DNA of C. pneumoniae, other bacteria or CMV identified in atheroma samples from small number of individuals. Contradicts most other findings and raises questions about sensitivity of methods used].]

Davidson, M., Kuo, C C., Middaugh, J. P., Campbell, L. A., Wang, S. P., Newman, W. P, III et al. (1998). Confirmed previous infection with Chlamydia pneumoniae (TWAR) and its presence in early coronary atherosclerosis. Circulation 98, 628 - 633. Full article   [An excellent and original study on 60 Native Alaskans at low risk of coronary heart disease. C. pneumoniae identified frequently in macrophage derived foam cells in plaque. High odds ratio for disease where IgG antibody to C. pneumoniae elevated. In some cases infection appears to precede early lesions].

Ericson, K., Saldeen, T. G., Lindquist, O., Pahlson, C. &  Mehta, J. L. (2000). Relationship of Chlamydia pneumoniae infection to severity of human coronary atherosclerosis. Circulation 101, 2568 - 2571. Full article [Presence of C. pneumoniae in post mortem tissue some subjects by immunocytochmistry correlates with severity of atherosclerosis. Partially contradicts Thomas et al., 1999, who used PCR and found no obvious relationship to severity].

Farsak, B., Yildirir, A, Akyon, Y., Pinar, A. Oc, M., Boke, E. et al. (2000). Detection of Chlamydia pneumoniae and Helicobacter pylori DNA in human atherosclerotic plaques by PCR. Journal of Clinical Microbiology 38, 4408 - 4411. [Presence of C. pneumoniae or H. pylori DNA associated with atherosclerosis] Full article

Jackson, L. A., Campbell, L. A., Kuo, C. C., Rodriguez, D. I., Lee, A. &  Grayston, J. T. (1997). Isolation of Chlamydia pneumoniae from a carotid endarterectomy specimen. Journal of Infectious Diseases  176, 292 - 295. [Viable C. pneumoniae are occasionally isolatable from arterial plaque]

Jantos, C. A., Nesseler, A., Waas, W., Baumgartner, W., Tillmanns, H. & Haberbosch, W. (1999). Low prevalence of Chlamydia pneumoniae in atherectomy specimens from patients with coronary heart disease. Clinical Infectious Diseases 28, 988 - 992. [Found low prevalence in coronary atheromas by PCR and in situ hybridization].

Kalayoglu, M. V., Libby, P. & Byrne, G. I. (2002). Chlamydia pneumoniae as an emerging risk factor in cardiovascular disease. JAMA. 288, 2724 - 2731. Full article

Kuo, C. C., Shor, A., Campbell, L. A., Fukushi, H., Patton, D. L. & Grayston, J. T. (1993). Demonstration of Chlamydia pneumoniae in atherosclerotic lesions of coronary arteries. Journal of Infectious Diseases 167, 841 - 849. [Follow up to Shor, Kuo & Patton 1992].

Kuo, C. C., Grayston, J. T., Campbell, L. A., Goo, Y. A., Wissler, R. W., Benditt, E. P. (1995). Chlamydia pneumoniae (TWAR) in coronary-arteries of young-adults (15- 34 years old). Proceedings of the National Academy of Sciences of the USA 92, 6911 - 6914. Full article    [Autopsy, formalin fixed tissue. C. pneumoniae present in atheromatous coronaries, not macroscopically normal ones]. 

LaBiche, R., Koziol, D., Quinn, T. C., Gaydos, C., Azhar, S., Ketron, G. et al. (2001). Presence of Chlamydia pneumoniae in human symptomatic and asymptomatic carotid atherosclerotic plaque. Stroke 32, 855 - 860.  [Careful NIH study. Presence of C. pneumoniae as a single factor does not appear to be sufficient to explain the occurrence of cerebrovascular symptoms].

Linares-Palomino, J., Gutierrez, J., Lopez-Espada, C., Ros, E., Moreno, J., Perez, T. et al. (2001).  Chlamydia pneumoniae and cerebrovascular disease. Revista De Neurologia 32, 201 - 206. [In Spanish. Small case control study].

Lindholt, J. S., Ostergard, L., Henneberg, E. W., Fasting, H. & Andersen, P.  (1998). Failure to demonstrate Chlamydia pneumoniae in symptomatic abdominal aortic aneurysms by a nested polymerase chain reaction (PCR). European Journal of Vascular and Endovascular Surgery 15, 161 - 164. [20 patients. Puzzling result].

Maas, M., Krause, E., Engel, P. M. & Kruger, S. (1997). Endovascular presence of Chlamydia pneumoniae in patients with hemodynamically effective carotid artery stenosis. Angiology 48, 699 - 706.

Ouchi, K., Fujii, B., Kanamoto, Y., Karita, M., Shirai, M. &  Nakazawa, T.(1998). Chlamydia pneumoniae in coronary and iliac arteries of Japanese patients with atherosclerotic cardiovascular diseases. Journal of Medical Microbiology 47, 907 - 913. [No control group].

Ouchi K., Fujii, B., Kudo, S., Shirai, M., Yamashita, K., Gondo, T. et al. (2000). Chlamydia pneumoniae in atherosclerotic and nonatherosclerotic tissue. Journal of Infectious Diseases 181 Suppl 3, S441 - S443. [Ch. pneumoniae found mainly in atherosclerotic tissue].

Ozsan, M., Gungor, C., Kahraman, M., Ozkul, A., Cinel, L., Tezcaner, T. et al. (2000). Chlamydia and atherosclerotic coronary arterial disease in Turkey. Acta Cardiology 55, 295 - 300. [Smallish study. No association presence of C. pneumoniae and disease or risk factors].

Paterson, D. L., Hall, J., Rasmussen, S. J. & Timms, P. (1998). Failure to detect Chlamydia pneumoniae in atherosclerotic plaques of Australian patients. Pathology 30, 169 - 172. [Autopsy study. Puzzling result. PCR inhibition not controlled.]

Petersen, E., Boman, J., Persson, K., Arnerlov, C., Wadell, G., Juto, P. et al. (1998). Chlamydia pneumoniae in human abdominal aortic aneurysms. European Journal of Vascular and Endovascular Surgery 15, 138 - 142. [C. pneumoniae present in high proportion of intraabdominal aneurysms. Contradicts Lindholt et al., 1998]. 

Radke, P. W., Merkelbach-Bruse, S., Messmer, B. J., vom Dahl, J., Dorge, H., Naami, A. et al. (2001). Infectious agents in coronary lesions obtained by endatherectomy: pattern of distribution, coinfection, and clinical findings. Coronary Artery Disease 12, 1 - 6. [C. pneumoniae and CMV DNA detected in approximately a third of coronary artery specimens. H. pylori DNA not detected. C. pneumoniae associated with unstable angina].

Shor, A., Kuo, C. C. & Patton, D. L. (1992). Detection of Chlamydia pneumoniae in coronary arterial fatty streaks and atheromatous plaques. South African Medical Journal 82, 158 - 161. [Classic paper of historic interest that kick started the direct search for the organism in CAD. Hallmark is the careful pathological observations of Alan Shor in S Africa. The first detection of C. pneumoniae in coronary arterial fatty streaks.]

Thomas, M., Wong, Y., Thomas, D., Ajaz, M., Tsang, V., Gallagher, P. J. et al. (1999). Relation between direct detection of Chlamydia pneumoniae DNA in human coronary arteries at postmortem examination and histological severity (Stary grading) of associated atherosclerotic plaque. Circulation 99, 2733 - 2766. Full article [The first study to look, within individuals, whether C. pneumoniae was associated with atherosclerosis, after correcting for the effects of PCR inhibition. No strong association found.]

Valassina, M., Migliorini, L., Sansoni, A., Sani, G., Corsaro, D., Cusi, M. G. et al. (2001). Search for Chlamydia pneumoniae genes and their expression in atherosclerotic plaques of carotid arteries. Journal of Medical Microbiology 50, 228 - 232. [Carotid endarterectomy specimens. Genomic DNA present, transcribed RNA not. Is this a sensitivity problem, or are C. pneumoniae present not metabolically active?]

Vink, A., Poppen, M., Schoneveld, A. H., Roholl, P. J. M., de Kleijn, D. P. V.,  Borst, C. et al. (2001). Distribution of Chlamydia pneumoniae in the human arterial system and its relation to the local amount of atherosclerosis within the individual. Circulation 103, 1613 - 1617. [Distribution of C. pneumoniae within an individual similar to that of atherosclerosis. Contradicts Thomas et al 1999, but using different techniques. Doesn't answer whether C. pneumoniae preferentially colonizes or causes atherosclerosis]. 

Weiss, S. M,. Roblin, P. M., Gaydos, C. A., Cummings, P., Patton, D. L., Schulhoff, N. et al. (1996). Failure to detect Chlamydia pneumoniae in coronary atheromas of patients undergoing atherectomy. Journal of Infectious Diseases 173, 957 - 962. [Small cross sectional study. Antibody to C. pneumoniae, unusually, higher in controls than patients]

Wong, Y. K., Dawkins, K. D. & Ward, M. E. (1999a). Circulating Chlamydia pneumoniae DNA as a predictor of coronary artery disease. Journal of the American College of Cardiology 34, 1435 - 1439. [Large study on 1300 patients indicating that the presence of circulating C. pneumoniae DNA in peripheral blood mononuclear cells was associated with greater risk of angiographic abnormalities in males]

Wong, Y. K., Gallagher, P. J. & Ward, M. E. (1999). Chlamydia pneumoniae and atherosclerosis. Heart 81, 232 - 238. [A critical review of the evidence that C. pneumoniae is an important cause of coronary heart disease. Found that although the majority of serological studies had shown an association between Ch pneumoniae and atherosclerosis, the number of cases in studies that have reported a positive association when using strict criteria for chronic infection is similar to the number of cases in studies which found no association.]

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