Chlamydialike organisms and atherosclerosis.

To the Editor: Chlamydophila pneumoniae causes pneumonia, but its role in the pathogenesis of atherosclerosis is controversial (1-4). The role of C. pneumoniae in atherosclerosis is supported by seroepidemiologic studies and detection in atherosclerotic lesions by polymerase chain reaction (PCR), immunohistologic analysis, culture, and electron microscopy (2,3). However, these results were not confirmed by other serologic or PCR-based studies (4). Meijer et al. evaluated abdominal aortic aneurysm biopsy specimens and detected C. pneumoniae membrane antigen more frequently than lipopolysaccharide antigens but did not detect heat shock protein 60 (1). In addition, they could not amplify or detect specific C. pneumoniae DNA by PCR and fluorescence in situ hybridization (1). They hypothesized that this discrepancy may result from a chlamydialike organism present in aortic samples that has surface antigens similar to those of C. pneumoniae.

Parachlamydia acanthamoebae and Neochlamydia hartmanellae are chlamydialike organisms that share [approximately equal to] 86% 16S rRNA sequence similarity with C. pneumoniae (5). Like C. pneumoniae, they have elementary and reticulate bodies visible by electron microscopy (6). Neochlamydia-related DNA (GenBank accession no. AF097191) has been amplified from 5 different arterial samples, including 1 aortic aneurysm (7), and a relationship (p = 0.009) between cerebral hemorrhage and serologic evidence of Parachlamydia infection has been reported (8). Therefore, we investigated the role of Parachlamydia in pathogenesis of atherosclerosis by using a molecular approach.

We analyzed 78 surgical samples from 27 patients undergoing aortic or carotid surgery for atherosclerotic disease at Hopital Nord in Marseille from June 1, 2003, to December 31, 2003. The study was approved by the local ethics committee, and written informed consent was obtained from all participants. Demographic and clinical data were prospectively recorded.

DNA was extracted from aortic or carotid samples with atherosclerotic lesions by using the QIAamp DNA tissue kit (Qiagen, Courtaboeuf, France), according to the manufacturer's instructions. A nested PCR was performed by using external primers 16SIGF (5'-CGGCGTGGATGAGGCAT-3') and 16SIGR (5'-TCAGTCCCAGTGTTGGC-3') (9) and internal primers CHL16SFOR2 (5'-CGTGGATGAGGCATGCAAGTCGA-3') and CHL16SREV2 (5'-CAATCTCTCAATCCGCCTAGACGTCTTAG-3') (7). PCR included negative controls from the DNA extraction step. DNA extractions and PCR amplifications were conducted in a laboratory in which parachlamydial DNA had not been extracted or amplified. PCR products were purified by using the Q1Aquick PCR purification kit (Qiagen) and sequenced by using the d-rhodamine terminator cycle sequencing reaction kit (Perkin-Elmer Biosystems, Warrington, UK) and a 3100 ABI Prism automated sequencer (Applied Biosystems, Courtaboeuf, France).

Sequences were analyzed with BLAST (http://www.ncbi.nlm.nih. gov/BLAST/) using gap existence and extension penalties of 5 and 2, respectively. Results were considered positive only when the sequence of the amplified product exhibited a best BLAST hit with a chlamydialike organism. Statistical analyses were performed with STATA software (Stata Corporation, College Station, TX, USA).

A positive PCR result was obtained with samples from 5 (18.5%) of 27 patients (Table). Three sequences had a best BLAST hit with the sequence of Parachlamydia sp. UV7 (GenBank accession no. A J715410), with a sequence similarity ranging from 99% to 100%. The other 2 sequences had 98% sequence similarity with Neochlamydia-related symbiont TUME-1 (GenBank accession no. AF098330). PCR positivity was not associated with age, sex, or location of the atherosclerotic lesion.

All patients with positive PCR results were [greater than or equal to] 68 years of age. Patients without cardiovascular risk factors were more likely than those with [greater than or equal to] 1 risk factor to have positive PCR results (p = 0.023). Despite the small number of patients in this study, this association was also confirmed in a multivariate logistic regression model adjusted for sex and previous cardiovascular disease (odds ratio 0.035, 95% confidence interval 0.001-0.94).

These findings suggest that Parachlamydia and Neochlamydia are associated with atherosclerosis. In addition, these obligate intracellular bacteria may be present in both carotid and aortic atherosclerotic lesions of elderly patients.

Chlamydialike organisms in atherosclerotic lesions may explain controversies about the role of C. pneumoniae in pathogenesis of atherosclerosis (4). Some PCRs might amplify both Chlamydia and chlamydialike organisms, leading to erroneous conclusions, especially when the specificity of the product is not confirmed by sequencing. Chlamydialike organisms in atherosclerotic lesions might also explain discrepancies of serologic studies (4). Parachlamydiaceae likely cross-react with C. pneumoniae (1). Such cross-reactivity will not be recognized if patients with positive serologic results for C. pneumoniae are not tested for antibodies to chlamydialike organisms. This cross-reactivity may result in false-positive serologic results for C. pneurnoniae, especially when low antibody titers (8-16) are considered positive.

Since elementary and reticulate bodies are similar in both Chlamydiaceae and Parachlamydiaceae (6), Chlamydiales cannot be identified in a specimen solely by electron microscopy. Consequently, elementary and reticulate bodies in atherosclerotic lesions (10) might be chlamydialike organisms and not C. pneumoniae. If chlamydialike organisms are involved in the pathogenesis of atherosclerosis, this finding would have public health implications, given their presence in free-living amebae that are widespread in water.

Acknowledgments

We thank Philip Tarr for reviewing the manuscript.

Dr Greub's research on chlamydialike organisms is currently supported by the Swiss National Science Foundation (SNSF) grant no. 3200BO-105885.

Gilbert Greub, * (1) Olivier Hartung, ([dagger]) Toidi Adekambi, * Yves S. Alimi, ([dagger]) and Didier Raoult *

* Universite de la Mediterannee, Marseille, France; and ([dagger]) Hopital Nord, Marseille, France

References

(1.) Meijer A, van der Vliet JA, Roholl PJ, Gielis-Proper SK, de Vries A, Ossewaarde JM. Chlamydia pneumoniae in abdominal aortic aneurysms: abundance of membrane components in the absence of heat shock protein 60 and DNA. Arterioscler Thromb Vasc Biol. 1999;19:2680-6.

(2.) Kalayoglu MV, Libby P, Byrne GI. Chlamydia pneumoniae as an emerging risk factor in cardiovascular disease. JAMA. 2002;288:2724-31.

(3.) Belland RJ, Ouellette SR Gieffers J, Byrne Gl. Chlamvdia pneumoniae and atherosclerosis. Cell Microbiol. 2004;6:117-27.

(4.) Ieven MM, Hoymans VY. Involvement of Chlamydia pneumoniae in atherosclerosis: more evidence for lack of evidence. J Clin Microbiol. 2005;43:19-24.

(5.) Greub G, Raoult D. Parachlamydiaceae: potential emerging pathogens. Emerg Infect Dis. 2002;8:625-30.

(6.) Greub G, Raoult D. Crescent bodies of Parachlamydia acanthamoeba and its life cycle within Acanthamoeba polyhaga: an electron micrograph study. Appl Environ Microbiol. 2002;68:3076-84.

(7.) Ossewaarde JM, Meijer A. Molecular evidence for the existence of additional members of the order Chlamydiales. Microbiology. 1999;145:411-7.

(8.) Greub G, Boyadjiev I, La Scola B, Raoult D, Martin C. Serological hint suggesting that Parachlamydiaceae are agents of pneumonia in polytraumatized intensive carc patients. Ann N Y Acad Sci. 2003;990: 311-9.

(9.) Everett KD, Bush RM, Andersen AA. Emended description of the order Chlamydiales, proposal of Parachlamydiaceae fam. nov. and Simkaniaceae fam. nov., each containing one monotypic genus, revised taxonomy of the family Chlamvdiaceae, including a new genus and five new species, and standards for the identification of organisms. Int J Syst Bacteriol. 1999;49:415-40.

(10.) Nystrom-Rosander C, Thelin S, Hjelm E, Lindquist O, Pahlson C, Friman G. High incidence of Chlamydia pneumoniae in sclerotic heart valves of patients undergoing aortic valve replacement. Scand J Infect Dis 1997;29:361-5.

(1) Current affiliation: University of Lausanne, Lausanne, Switzerland

Address for correspondence: Gilbert Greub, Center for Research on Intracellular Bacteria, Microbiology Institute, Faculty of Biology and Medicine, University of Lausanne, 1011 Lausanne, Switzerland; fax: [phone]; email: [e-mail]

Table. Comparison of patients with and without parachlamydial DNA by
polymerase chain reaction (PCR) in surgical aortic or carotid specimens

                                Positive PCR    Negative PCR
                                   result          result
Characteristic                    (n = 5)         (n = 22)      p value

Males, no. (%)                    4 (80)         16 (73)         0.74
Median age (interquartile
  range)                         72 (71-77)      66 (61-74)      0.11
Any cardiovascular risk
  factor, no. (%)                 3 (60)         21 (95)         0.02
  Hypertension                    3 (60)         10 (45)         0.56
  Hypercholesterolemia               0            5 (23)         0.24
  Tobacco use                     3 (60)         17 (77)         0.42
  Diabetes                        1 (20)          6 (27)         0.74
Aortic surgery, no. (%)           2 (40)         13 (59)         0.44
Previous cardiovascular
  disease, no. (%)                2 (40)          8 (38)         0.94