*2.1.1 Production in culture*

Ramirez et al. [23] reported the first case of *C. pneumoniae* that could be isolated from a coronary artery plaque and cultured in vitro in 1996. Jackson et al [24].

demonstrated the presence of *Chlamydia* by immunohistochemical, PCR, or electron microscopy in 75% of 25 patients with carotid endarterectomy, whereas culture was positive in only one patient. A small proportion of atherosclerotic plaques with *Chlamydia* presence confirmed using other diagnostic methods have been successfully cultured [25, 26]. Karlsson et al. [27] demonstrated the presence of *C. pneumoniae* immunohistochemically in 20 of 26 abdominal aortic aneurysm tissue specimens, but were able to isolate *Chlamydia* in culture media in 10 cases. *Chlamydia* are reported to be difficult bacteria to culture because of their biphasic life cycle. It is accepted that in vitro culture has low sensitivity in demonstrating the presence of *Chlamydia* in tissue examinations [28].

#### *2.1.2 Serological investigations*

While some of the studies using serological tests showed a positive relationship between *C. pneumoniae* infection and coronary atherosclerosis, some studies reported that this relationship could not be accepted as sufficient evidence for the etiology of atherosclerosis [29–31].

Some studies revealed a relationship between *C. pneumoniae*-specific IgG and IgA positivity and atherosclerosis development, whereas other researchers did not observe this relationship [32–35]. Serological tests are based on the detection of anti-chlamydial antibodies (IgA, IgG) in blood samples. However, these antibodies are indicators of the immune response rather than active infection. They also show cross-reactivity with other *Chlamydia* species. Danesh et al. [36] conducted a metaanalysis of 14 prospective studies including a total of 3619 patients and reported that there was no relationship between *C. pneumoniae* antibodies and atherosclerotic heart disease. This is similar to the presence of antituberculosis antibodies in serological tests after tuberculosis vaccination. The presence of antibodies may not mean there is active infection. In addition, it was shown that serological tests were negative even though the presence of *Chlamydia* could be demonstrated in the atherosclerotic plaques of immunodeficient individuals [19]. In general, pathogenic processes that cause an inflammatory response (such as smoke exposure, hypertension, hyperlipidemia, malignancy, and hyperglycemia) are known to cause errors in serological tests, which reduces their specificity. As a result, it is accepted that serological studies are not useful and are insufficient in the detection of *Chlamydia*.

#### *2.1.3 Techniques for demonstrating* Chlamydia *in atheromatous plaques*

Although most studies conducted in different centers in many parts of the world were able to demonstrate the presence of *Chlamydia* in atherosclerotic lesions, they could not be detected in other studies. This was thought to be related to differences or technical incompatibilities between the imaging methods, leading to debate regarding which diagnostic methods are most appropriate. As a result, polymerase chain reaction (PCR), immunohistochemistry (microimmunofluorescence), and electron microscopy imaging are the most widely accepted techniques. These studies focus on the bacteria's DNA signature or directly demonstrate bacterial presence instead of utilizing indirect methods.

PCR is a diagnostic method for detecting chlamydial DNA or RNA and focuses on the bacteria's genetic material [37]. It is a sensitive and specific test based on the degradation of genetic material in atheromatous plaques by electrophoresis [38, 39]. Immunogold labeling is based on the direct observation of *Chlamydia* bacteria with

an electron microscope. The detection of monoclonal antibodies clearly demonstrates the presence of *Chlamydia*. Immunocytochemistry (ICC) is based on the demonstration of anti-chlamydial immunoglobulins adhering to the *Chlamydia* bacteria using immunofluorescent microscopy. This method has lower specificity and sensitivity than PCR. Serologic studies are based on the measurement of the host response to chlamydial invasion. Results are obtained by demonstrating host immunoglobulins. It has low specificity and sensitivity.

In most studies using ICC, PCR, and culture methods for *C. pneumoniae*, detection rates were higher in atherosclerotic vessels than in those without atherosclerosis [40, 41].
