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.

Keywords: Chlamydia pneumoniae; PCR; acute coronary syndrome; cultured cells; human serum.

Figure 1

Recovery of PCR product in DNA samples isolated from serum specimens using QIAamp DNA blood midi kit, protein A and phenol-chloroform extraction method. 1 - molecular size standards; 2, 6 and 10 - PCR-positive serum from patient M; 3, 7 and 11 - PCR-positive serum from patient P; 4, 8 and 12 - PCR negative serum from patient S; 5, 9 and 13 - extraction control; 14 - negative control; 15 - positive control.

Figure 2

Recovery of PCR products in amplification reactions with different primers (chlamydial 16 S rRNA and omp1) using DNA extracted from human plasma by phenol-chloroform method. 1 - molecular size standards; 2, and 8 - PCR positive serum from patient M.; 3 and 9 - PCR positive serum from patient P.; 4 and 10 - PCR negative serum from patient S.; 5 and 11 - extraction control; 6 and 12 - negative controls; 7 and 13 - positive controls.

Figure 3

Electron-microscopic images of C.pneumoniae elementary bodies obtained from HL cells infected with C. pneumoniae reference strain (A and B) and serum centrifugates (C and D).

Figure 4

Electron-microscopic images. Immunogold labeling of C.pneumoniae elementary bodies in serum sediments. A - preincubation with normal mouse IgG. B - preincubation with monoclonal antibody against chlamydial LPS.

Figure 5

Immunofluoresence analysis of HL monolayers after inoculation of serum sediments (A), and reference culture (B) of C. pneumoniae.