Higher incidence of persistent chronic infection of Chlamydia pneumoniae among coronary artery disease patients in India is a cause of concern

Abstract

Background: There is growing evidence that Chlamydia pneumoniae may be involved in the pathogenesis of atherosclerosis, as several studies have demonstrated the presence of the organism in atherosclerotic lesions. C. pneumoniae infections, which are especially persistent infections, have been difficult to diagnose either by serological methods or isolation of the organism from the tissue. Nucleic Acid Amplification tests (NAATs) has emerged as an important method for detecting C. pneumoniae. Inspite of high prevalence of C. pneumoniae specific antibodies in coronary heart disease patients, direct detection of C. pneumoniae in circulating blood of coronary artery disease (CAD) patients by sensitive nucleic acid amplification tests nested PCR (nPCR), multiplex PCR (mPCR) has not been carried out is required. Further correlation of the presence of C. pneumoniae in blood of CAD patients with C. pneumoniae specific IgA and IgG antibodies, which may indicative of the status of infection with the progression of atherosclerosis. This will help in order to prepare strategies for the antibiotic intervention to avoid the progression towards CAD.

Methods: Venous blood was obtained from 91 CAD patients and 46 healthy controls. Nucleic acid amplification tests viz. nested-, semi-nested- and multiplex PCR were used for detection of C. pneumoniae. ELISA carried out prevalence of C. pneumoniae specific IgG and IgA antibodies.

Results: 29.67% (27/91) patients were positive for C. pneumoniae using nested PCR. The sensitivity and specificity of semi-nested and multiplex PCR were 37.03%, 96.96% and 22.22%, 100% with respect to nested PCR. Positive nPCR patients were compared with presence of C. pneumoniae specific IgA, IgA+IgG and IgG antibodies. Among 27 (29.67%) nPCR C. pneumoniae positive CAD patients, 11(12%) were IgA positive, 13(14.2%) were IgA+IgG positive and only1 (1.1%) was IgG positive. A significant presence of C. pneumoniae was detected in heavy smokers, non-alcoholics and with family histories of diabetes and blood pressure group of CAD patients by nPCR.

Conclusion: The results indicate synergistic association of C. pneumoniae infection and development of CAD with other risk factors. We also detected increased positivity for C. pneumoniae IgA than IgG in nPCR positive CAD patients. Positive nPCR findings in conjunction with persisting high C. pneumoniae specific antibody strongly suggest an ongoing infection.

Figure 1

mPCR for 16SrRNA C. pneumoniae specific gene. Amplified product was electrophoresed on 1.2% agarose gel yielding a 463 bp product. Lane 1 Positive control, Lane 2, 4, 5 DNA of CAD patients, Lane 3 negative control and Lane 6, 100 bp ladder.

Figure 2

snPCR for Omp1 C. pneumoniae specific gene. Amplified product was electrophoresed on 1.5% agarose gel yielding a 360 bp product. Lane1 positive control, Lane 2&3 DNA of CAD patients, Lane 4, 100 bp ladder, Lane 5, negative control.

Figure 3

nPCR for 16SrRNA gene specific for C. pneumoniae. Amplified product was electrophoresed on 1.2 % agarose gel yielding a 570 bp product. Lane 1 Negative control, Lane 2 100 bp ladder Lane 3 positive control, Lane 4, 5, 6, CAD patients DNA

Figure 4

Quantitative nPCR for 16SrRNA gene specific for C. pneumoniae. Amplified product was electrophoresed on 1.2 % agarose gel yielding a 570 bp product. Lane 1 100 bp ladder, Lane 2 negative control, Lane 3,4,5 and 6, CAD patients DNA 100, 75, 50, 25 ng respectively.

Figure 5

Graph showing % positivity of C. pneumoniae in CAD patients with different smoking habits. On Y axis % positivity for C. pneumoniae positivity is depicted. On X-axis; smoker groups are divided into heavy smoker, occasional smoker and never smoker.

Figure 6

Graph showing % positivity of C. pneumoniae in CAD patients with different alcohol consumption. On Y axis % positivity for C. pneumoniae is depicted. On X-axis- alcoholic groups divided into heavy alcoholic, occasional alcoholic and never taken alcohol.

Figure 7

Graph showing percentage of C. pneumoniae positivity in CAD patients with serology group IgA, IgA+IgG, IgG and with nPCR. On Y axis % positivity in CAD patient for C. pneumoniae is depicted. On X-axis serological marker IgA, IgA+IgG, IgG with nPCR.