Chlamydia pneumoniae, Stroke, and Serological Associations
Anything Learned From the Atherosclerosis-Cardiovascular Literature or Do We Have to Start Over Again?
There are now more arguments against than for a causal relationship between C. pneumoniae and atherosclerosis. Seroepidemiologic results are largely technique-dependent; PCR results show intra- and interlaboratory variability; methodological factors contribute to bias; and detection of C. pneumoniae fails when the specificity of the reaction is optimized. Immunohistochemical staining detects nonspecific compounds in atherosclerotic lesions, and secondary prevention trials are unsuccessful. The evidence for an association between a pathogen and a chronic disease should be based on concordance of evidence arising from different approaches applied by different groups, at different times, in different places, and under different circumstances. None of these conditions have been fulfilled in the case of C. pneumoniae and atherosclerosis.1
Citing the concluding paragraph of Margareta Ievens’ excellent review on Chlamydia pneumoniae and atherosclerosis, published in 2005 by the American Society of Micobiology (ASM) in their flagship journal for clinical microbiologists, the Journal of Clinical Microbiology, probably best summarizes the problems arising from diagnostic shortcomings of C pneumoniae, a microorganism that has been associated with well over 30 various diseases of markedly different pathologies and pathogenic mechanisms. Even though there is now “more evidence of no evidence” of the particular association on “C pneumoniae and atherosclerosis” than ever, every month articles continue to be published based on methods for which there is evidence that they are inadequate at various levels to define a patients’ C pneumoniae status. C pneumoniae serology is one of these methods.
In this issue of Stroke, Elkind et al2 studied whether evidence of C pneumoniae infection is associated with risk of first ischemic stroke. Again, a study based on serology, using the microimmunofluorescence technique (MIF), found elevated C pneumoniae IgA titers were associated with in-creased risk of first ischemic stroke after adjusting for conventional risk factors. The authors also found a trend toward an association of elevated IgA titers in atherosclerotic and lacunar stroke types. They concluded that IgA may be a better marker of risk than IgG and that further studies of the effect of C pneumoniae on stroke risk are warranted. In the light of what we know already from the cardiovascular and microbiological literature, are there indeed more studies on this association warranted?
The major flaws of this otherwise well-designed study by Elkind et al2 are 2-fold. First, the authors deal with the parameter “IgA against C pneumoniae detected by MIF” as if it were the same as having measured cholesterol, C-reactive protein or any other parameter, for the measurement of which a standardized, validated test system exists. Second, the authors assume a single IgA MIF titer of ≥16 being diagnostic for chronic, persistant C pneumoniae infection.
I would like to address the second issue first. If the half-life of C pneumoniae-specific IgA is only a few days, where is the focus of the assumed persistant infection? C pneumoniae is thought to be a respiratory pathogen. Is it the respiratory tract, steadily triggering IgA production leading to large vessel atherosclerotic and lacunar stroke types? The authors did not address the issue of prior respiratory tract infections. In their discussion they comment that they had no data to address this issue. Initially, however, they looked at oropharyngeal swabs with culture and polymerase chain reaction (PCR). What was that done for, if not to study respiratory tract colonization/infection? Because of the very low yield of these techniques, they subsequently discontinued direct detection of C pneumoniae. The discrepany between serology and direct detection in the study by Elkind et al2 could be another good example that detection of C pneumoniae fails when the specificity of the reaction is optimized.
If not the respiratory tract, is it then the assumed “vascular” chlamydial infection, steadily triggering IgA production? Affected vascular tissue was not studied by Elkind et al.2 Even if they had studied cerebrovascular atherosclerotic lesions by direct detection methods it would have been difficult to interpret their findings and compare them to currently published data. To illustrate the diagnostic dilemma: as of 2005, 28 out of 38 peer-reviewed studies addressed the question of whether C pneumoniae-DNA is present in cerebrovascular atherosclerotic tissues by PCR. Data on 971 atherosclerotic carotid arteries and 731 blood specimens from 1485 patients analyzed by 35 in-house PCR protocols found positivity ratios between 0% and 100%, apparently even when the same methods were used. No 2 studies were comparable in terms of methodology applied.3 There can be problems with sensitivity, specificity, and contamination, which can be secondary to a very large number of technical issues. The list of potential pitfalls is long, and failure to correctly address a single point might lead to loss of sensitivity but also to serious specificity problems, resulting in flawed results followed by misleading conclusions and biased associations.4
To come back and address the first issue as objected above: C pneumoniae is thought to be a rare cause of community-acquired pneumonia. Not even in this indication is it clear, in general, how to make the diagnosis, and, in particular, how to define a patient experiencing “chronic” infection. Because C pneumoniae is an intracellular pathogen, it is not surprising that there is a poor correlation between direct detection (eg, by culture and/or PCR) and serology. Whether serology might be a useful diagnostic tool to diagnose C pneumoniae infection, and if yes, which classes and titers of antibodies might represent acute first or reinfection, chronic, persistent or past C pneumoniae-infection is, unfortunately, not clear at all. To give an example of the complexity of this issue: 2 multicenter pneumonia treatment studies showed that although 7% to 13% of the patients in the study were culture positive, and 7% to 18% met the serologic criteria5 with the MIF for acute infection, they were not the same patients. Only 1% to 3% of the culture positive patients met the serologic criteria and ≈70% with positive cultures for C pneumoniae were seronegative.5 Another problem with serologic diagnosis of C pneumoniae infection is that the MIF method used to detect serum antibodies is not standardized, and recent studies have shown that there is substantial interlaboratory variation in the performance of these tests.6 So, again, what does a single IgA MIF titer of ≥16 mean?
If C pneumoniae serology is problematic in making the diagnosis of a disease to which the pathogen is most affiliated to—community acquired pneumonia—is it justified to apply such a test for clinical research purposes? The fact that cell culture for isolation is fastidious and that there is no standardized and validated molecular method available makes serology for C pneumoniae neither a better diagnostic option nor a reliable research tool, especially not if the Chlamydial status of patients presenting with various types of stroke or controls has to be defined.
Whatever was measured (or not) in the various stroke subgroups in the study by Elkind et al,2 the specifiticy for C pneumoniae has to be questioned.
Nearly 20 years ago, Saikku et al7 reported that patients with coronary artery disease were significantly more often positive for anti-C pneumoniae IgG and IgA than healthy controls. Later prospective studies, in which results were generally adjusted for traditional risk factors, seemed to minimize the relationship between baseline C pneumoniae titers in the healthy population and the risk for a subsequent coronary event. Furthermore, the presence of elevated anti-C pneumoniae antibodies in patients with preexisting vascu-lar disease meant no increased risk for future or recurrent cardiovascular events. Is it plausible that in cerebrovascular atherosclerosis things would be completely different to what it is like in cardiovascular atherosclerosis? On the other hand, if it is not specific immunoglobulin directed against C pneumoniae, what then is it, that has been measured by MIF tests in so many studies, and lately in that by Elkind et al2?
A serological test can only be as specific as the antigen used. Cross-reactivity between C pneumoniae and other Chlamydia species has been demonstrated with the MIF test. Factors like the strain type, purity, and concentration of the antigen used and the assay procedure itself might contribute to the fact that the MIF is less specific for C pneumoniae as thought 20 years ago.
Chlamydiae are a unique group of obligate intracellular bacteria comprising important pathogens of vertebrates as well as symbionts of free-living amoebae. In this context it is important to know that new environmental Chlamydia species are steadily described.8,9⇓ There is ample evidence for a huge diversity and wide distribution of Chlamydiae in nature—and we are exposed to that diversity of species. For example, until recently the recovery of a novel environmental Chlamydia strain from activated sludge by cocultivation with Acanthamoeba species was reported,8 and it was shown that it is also able to invade mammalian cells. These new environmental Chlamydiae (ie, Simkania, Waddlia and Parachlamydia) may significantly interfere with diagnostic testing for traditional Chlamydiaceae (ie, Chlamydophila and Chlamydia).
In summary, C pneumoniae serology is most problematic in terms of specificity, reproducibility, and what titer, even if prospectively defined, may mean what in a given clinical picture or disease. The specificity for C pneumoniae of the MIF, the serologic “golden standard”, may have been largely overestimated in the past. Besides, there are newer data which also show how subjective interpretation and intra- as well as interlaboratory reproducibility are. The problems in context with C pneumoniae serology (also if MIF based) have been discussed in detail in 2 recently published review articles.1,10⇓ Even if these articles have focused on cardiovascular atherosclerosis, the methodological issues remain the same in cerebrovascular atherosclerosis.
For the reasons outlined, not a single test, neither molecular biology-based ones nor serology, including the “golden standard” MIF, has achieved approval by the US Food and Drug Administration. The findings by Elkind et al2 as well as similar studies have to be read in that context.
Otherwise, I would like to leave it to both reviewers and readers alike whether or not we do need more studies on the association of C pneumoniae and various stroke types, or whether we should wait unless we know better how to accurately determine a patients’ C pneumoniae status.
The opinions in this editorial do not necessarily reflect those of the editors or of the American Heart Association.
See related article, pages 790–795
Ieven MM, Hoymans VY. Involvement of Chlamydia pneumoniae in Atherosclerosis: More Evidence for Lack of Evidence. J Clin Microbiol. 2005; 43: 19–24.
Elkind MSV, Tondella MLC, Feikin DR, Fields BS, Homma S, Di Tullio MR. Seropositivity to Chlamydia pneumoniae is associated with risk of first ischemic stroke. Stroke. 2006; 37: 790–795.
Apfalter et al. 2006, Chlamydia pneumoniae, cerebrovascular atherosclerosis and the critical impact of nucleic acid amplification technology. Current Medicinal Chemistry, invited review, in press.
Apfalter P, Reischl U, Hammerschlag MR. In-house nucleic acid am-plification assays in research: how much quality control is needed be-fore one can rely upon the results? J Clin Microbiol. 2005; 43: 5835– 5841.
Hammerschlag MR. Chlamydia pneumoniae and the lung. Eur Resp J. 2000; 16: 1001–1007.
Collingro A, Poppert S, Heinz E, Schmitz-Esser S, Essig A, Schweikert M, Wagner M, Horn M. Recovery of an environmental Chlamydia strain from activated sludge by cocultivation with Acanthamoeba sp. Microbiology. 2005; 151: 301–309.
Griffiths E, Petrich AK, Gupta RS. Conserved indels in essential proteins that are distinctive characteristics of Chlamydiales and provide novel means for their identification. Microbiology. 2005; 151: 2647–2657.
Boman J, Hammerschlag MR. Chlamydia pneumoniae and Atherosclerosis: Critical Assessment of Diagnostic Methods and Relevance to Treatment Studies. Clin Microbiol Rev. 2002; 15: 1–20.