ISOLATION OF CHLAMYDIA PNEUMONIAE FROM SERUM SAMPLES OF THE PATIENTS WITH ACUTE CORONARY SYNDROME

Abstract

BACKGROUND: Limited body of evidence suggests that lipopolysaccharide of C. pneumoniae as well as C. pneumoniae-specific immune complexes can be detected and isolated from human serum. The aim of this study was to investigate the presence of viable elementary bodies of C.pneumoniae in serum samples of patients with acute coronary syndrome and healthy volunteers.MATERIAL AND METHODS: Serum specimens from 26 healthy volunteers and 56 patients with acute coronary syndrome were examined subsequently by serological (C.pneumoniae-specific IgA and IgG), PCR-based and bacteriological methods. Conventional, nested and TaqMan PCR were used to detect C.pneumoniae genetic markers (ompA and 16S rRNA) in DNA from serum specimens extracted with different methods. An alternative protocol which included culturing high-speed serum sediments in HL cells and further C.pneumoniae growth evaluation with immunofluorescence analysis and TaqMan PCR was established. Pellet fraction of PCR-positive serum specimens was also examined by immunoelectron microscopy.RESULTS: Best efficiency of final PCR product recovery from serum specimens has been shown with specific C. pneumoniae primers using phenol-chloroform DNA extraction protocol. TaqMan PCR analysis revealed that human serum of patients with acute coronary syndrome may contain genetic markers of C. pneumoniae with bacterial load range from 200 to 2000 copies/ml serum. However, reliability and reproducibility of TaqMan PCR were poor for serum specimens with low bacterial copy number (<200 /ml). Combination of bacteriological, immunofluorescence and PCR- based protocols applied for the evaluating HL cells infected with serum sediments revealed that 21.0 % of the patients with acute coronary syndrome have viable forms C.pneumoniae in serum. The detection rate of C.pneumoniae in healthy volunteers was much lower (7.7%). Immunological profile of the patients did not match accurately C.pneumoniae detection rate in serum specimens. Elementary bodies of C.pneumoniae with typical ultrastructural characteristics were also identified in serum sediments using immunoelectron microscopy.Conclusions: Viable forms C. pneumoniae with typical electron microscopic structure can be identified and isolated from serum specimens of the patients with acute coronary syndrome and some healthy volunteers. Increased detection rate of C. pneumoniae in serum among the patients with an acute coronary syndrome may contribute towards enhanced pro-inflammatory status in cardiovascular patients and development of secondary complications of atherosclerosis.

Chlamydia, Hepatocytes and Liver

“…Furthermore, multiple surface-located adhesins, represented by the family of polymorphic membrane proteins (Molleken et al, 2010), are known to promote the adherence of chlamydial biovars to the endothelial cell and lymphocytes . However, according to our recently published results, EB of C. pneumoniae are identifiable by both electron microscopy and RT-PCR in serum specimens obtained from the patients with acute coronary syndrome (Petyaev et al, 2010). By a conservative estimate, our finding may be attributed to the partial lysis of blood cells during serum isolation.…”

“…Moreover, liver is known to express abundantly LDL-receptor (Dietschy & Turley, 2002), which can be implemented in our view in the pathogenesis of chlamydiosis. Using an immunoprecipitation approach we have found (Petyaev et al, 2010) that two chlamydial biovars -C. trachomatis and C. pneumoniae can bind ApoB-containing lipoproteins under "in vitro" conditions. This was strikingly distinct from no interaction with the HDL fraction.…”

“…This difficulty can be partially overcome by the use of freshly isolated hepatocytes plated onto collagen-coated dishes in the presence of certain hormones (Shimomura et al, 1999). It has been shown during the last few years that major chlamydial species can efficiently propagate in cultured hepatoma cells (Wang et al, 2007;Bashmakov et al, 2010) and accomplish their entire developmental cycle with the final release of infective progeny from ruptured hepatocytes. A similar observation has been made in experiments with freshly isolated hepatocytes from rat liver (I.M .…”

Selective Pressure Promotes Tetracycline Resistance of Chlamydia Suis in Fattening Pigs

“…Petayaev et al [25] showed that as few as 200 copies per ml by quantitative TaqMan PCR were enough to successfully isolate C . pneumoniae from the sera of patients with acute coronary syndrome.…”

Lack of strong association of Chlamydia pneumoniae and atherosclerosis in a Jordanian population

“…In these studies, the prevalence ranged from 4%-87% in patients and from 0%-50% in controls [10,14]. In addition to the data published above on PBMCs testing, Petyaev et al successfully confirmed the presence of C. pneumoniae by culturing methods and PCR in serum samples of patients with acute coronary syndromes [40]. Here, we tried to demonstrate C. pneumoniae in the bloodstream by isolating the bacterial DNA from the whole blood.…”