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Indian Journal of Community Medicine

Evaluation of Antichlamydia Pneumonia Antibody in Patients With Coronary Artery Disease

Author(s): M. Karimi, H. Nough, M.H. Lotfi , A.J. Zadeh, J. Ahmadi, M.S. Fathollah, M. R. Behforooz

Vol. 31, No. 4 (2006-10 - 2006-12)

M. Karimi, H. Nough, M.H. Lotfi , A.J. Zadeh, J. Ahmadi, M.S. Fathollah, M. R. Behforooz

Abstract:

Research Question: is there any mean difference in anti-Chlamydia Pneumonia (CP) IgG antibody between patients with Coronary artery events and healthy subjects? Objective: To compare mean of anti-Chlamidya IgG antibody amongst patients with Acute Myocardial Infarction (MI), Chronic Stable Angina (CSA) and Healthy persons. Study Design: A hospital based case-control study during Dec.2003 to Mar. 2004. Setting: A hospital based study in the preventive cardiology clinic of Aliebne- Abitaleb hospital, Rafsanjan city, Iran. Participants: A total 90 subjects, 30 subjects with acute MI, 30 with CSA and 30 healthy subjects. Statistical Analysis: non-parametric tests (chi-square test, Kruscal and Alis and Dunn test). Results: Mean antibody levels against CP in acute MI; chronic stable angina and control subjects were 34.7, 11.4 and 3,6 u/ml respectively. Obtained differences were found to be statistically significant. (P<.001). Conclusion: The results showed that there was an increased level of serum anti- CP antibody in higher percentage of CAD patients compared to control subjects and so expected to be a significant predictor of getting CAD.

Key words: Antibody, Chlamydia Pneumonia (CP), Coronary Artery Disease (CAD), Myocardial Infarction (MI).

Introduction

Atherosclerosis is the main cause of cardio-vascular diseases. Framingham’s Heart Study and other studies have showed main risk factors of CAD including hypertension, hypercholesterolemia, diabetes, smoking, environmental factors (such as unsuitable diets, less physical activity) and hereditary factors1-3. The role of infection as risk factors of atherosclerotic diseases has a 100- year record but the first published texts related to the atherogenic role of virus appeared in 1970. At present, several infectious agents resulting in chronic infections have been specified as probable contributor in creation of atherosclorosis.4-7

According to “response to damage” hypothesis, the atherosclerosis damage will initiate the additional response including infl ammation and reproduction of fibro connective tissues8. Different chronic CP (Chlamydia pneumonia) and dental infections are considered as risk factors of brain strokes, MI and atherosclorosis. At present, the strong evidences show that CP causes atherosclorosis. Serologic findings including isolated CP in atheroma plaques, experimental evidence and studies through which an antibiotic is used against CP especially chronic ones, suggest that infection created by this organism is considered as strong risk factor in causing atherosclorosis disease10,11. At the moment, it is not clear whether CP infection is primary contributor in the first phase of atherosclorosis or it account for secondary atheroma plaque. Age, onset of primary CP infection, smoking and genetic factors are determinant factors of chronic CP infections and they along with nutritional factors different societies. Otherwise several large scale prospective studies have not found evidence that prior exposure to CP is associated with increased risk of future cardiovascular events. Furthermore, recovery of infections particles within atheromatous plaque does not prove causation but may simply represent an innocent commensal colonization. It is thus uncertain whether infection plays an important etiologic role in atherothrombosis.23,24 Other infections such as Helicobacter, Herpes virus and HIV virus play a role in pathogensis of atherosclorosis.14,15

To determine the probable role chlamydia pneumonia in atherosclorosis, we arranged a study on the level of anti CP antibody in patients with having complication of coronary atherosclorosis such as acute MI and chronic stable angina.

Material and Methods

This The study aimed to evaluate and quantify the level of anti-Chlamidya pneumonia antibody in the serum of subjects with Coronary artery events by through a case-control study. The study was done in Ali-ebne Abitaleb hospital affi liated to Rafsanjan University of Medical Sciences, The Islamic Republic of Iran from December 2003 to March 2004. A total sample size of 60 was calculated using appropriate formula; N= [2(zα / 2 + z (1-Β)2*P* q] / (pl- p2)2 with (α= .05 & Β = .20); based on specified proportion of CAD cases (p1=50%) and controls (p2=16%)7,17 with anti-Chlamydia pneumonia antibody. The main purpose of study was to compare the amount of anti-Chlamydia pneumonia antibody in MI cases and control subjects. However, to know the antibody status in patients with Angina Pectoris, we selected 30 more subjects suffering from chronic stable angina without any history of acute MI and compared them with MI cases and the same controls. After making sure that MI patients were stable after acute MI and also other subjects were comfortable and felt geared up to answer the questions and taking blood sample, a written consent was obtained for the purpose of ethical clearance. Then the subjects were classified in three groups:

Group 1: 30 Patients of 40- 65 years old age with definite acute MI diagnosed based on criteria as per the MONICA project – 1) two or more ECG showing specific changes, 2) ECG showing probable changes plus abnormal cardiac injury enzymes, or 3) Typical symptoms such as retrosternal pain plus abnormal enzymes. Acute MI cases confirmed by expert cardiologist were selected from Coronary Care Unit ward of Ali-ebne Abitaleb hospital. Group 2: 30 Patients suffering from chronic stable angina without any history of acute MI and confirmed by expert cardiologist were selected from patients referred to OPD ward of Hospital. Group 3: 30 healthy persons without any sign and symptoms of coronary artery disease were selected as control group according to an initial analysis of residence place of MI cases. Patients having chronic bronchitis and patients being cured by macrolidl antibiotics were excluded from the study.

Sex, age and residence place of different groups were matched. After selecting the samples, we first numbered them from 1 to 30, and then a sample of 3ml venous blood was taken from each person in a sterile condition. Later, collected samples were centrifuged for 4 minutes (1-2 hours after sampling with speed of 3000 rounds per minute) and the serum of samples were isolated and freezed in -20°c. Finally, the level of IgG antibody in samples (antichlamydia pneumonia) were determined through by Eliza method and 96 test kite (Enzyme Immunoassay). The method of measuring IgG antibody to antichlamydia pneumonia: At the first we added 100 microlitre of serum sample of subjects and 100 microlitre of the standard serum individually to the microplate holes bottom of which have been already covered with antigenes of outside membrane of CP. Immediately 100 microlitre of anti- Human IgG which “Labelled by aperoxidase Enzyme” were added to the holes. Then we covered the microplate by plastic cover and incubated it in 37°c for one hour. After passing incubation time, the residual solution in the microplate hole were left away and then holes were washed by standard washing solution for 4 to 5 times. Then 100 microlitre chromogen tetra Ethyl benzydine was added to the holes and incubated for 20 minutes in darkness and laboratory temperature. When the time of incubation overed, the reaction was stopped by 100cc the stopping solution (Acid sulphoric 0.5 normal), then tile strength of color (od) of samples were read by Eliza in wave length of 450 nanometer. Based on data on the kites, the standard diagram was drawn and level of antichlamydia antibody was determined. The amounts of antibody more than 5 unit in ml were considered as positive. Sensitivity and specifi city of methods used were 98% and hemolped samples were excluded from the study (from each group one sample was omitted). All samples were done by a laboratory specialist. Having determined the level of IgG anti-CP antibody in the samples, the data was analyzed in Instant software program by using non-parametric tests (chi-square test, Kruscal and Alis and Dunn test) to compare the relations among 3 groups. A significance level of .05 was set for the interpretation of possible significant findings.

Results

Mean age in three groups was 52 ± 8. There was in each group 18 men (62%) and 11 women (38%). The percentages of positive serum antibody (the level of IgG anti-CP antibody more than 5 unit/ml) in groups 1, 2, and 3 were 100%, 65.6% and 31% respectively The association of level of anti- CP antibody with CAD was found to be statistically significant. (X2 = 30.526 and P<0.05) (Table 1). The mean concentration of IgG anti- CP antibody in groups of 1, 2 and 3 was 34.7± 4, 11.4 ± 2.1 and 3.6 ± 0.6-unit/ ml respectively that the differences were shown to be statistically significant (kw value= 51.58 and p= 0.001) (Table 2). The mean concentration of IgG antibody in the men of groups of 1, 2 and 3 was 37.4± 5.9, 13.6 ± 3.2 and 3.1 ± 0.6 unit/ ml respectively so that mean differences between groups 1 and 2 and 2 and 3, also between groups 1 and 3 were statistically significant (p< 0.01). However, the mean differences between female subjects of groups 2 and 3 were not found to be statistically significant (p> 0.05).

Table I. The Frequency of lgG Anti-Chlamydia Pneumonia Antibody in the Studied Groups.

Group Positive
(IgG>5 u/ml)
No. %
Negative
(IgG <5u/ml)
No. %
Total
1. Acute MI 29(100) 0 (0) 29(33.33)
2. Chronic
stable angina
19(65.6) 10(34.4) 29(33.33)
3. Control 9(31) 20(69) 29(33.33)
  Total 57(65.6) 30(34.4) 87(100)

X2 = 30.526 P- value < 0.0001

Table II. Mean comparison of IgG anti CP antibody in the studied groups.

Group No Minimum
(u/ml) IgG
Maximum
(u/ml) IgG
Mean ± SEM
1. Acute MI 29 7 85.13 34.7± 4
2. Chronic
stable angina
29 0.5 48.5 11.4± 2.1
3. Control 29 0.25 12.5 3.6±0.6

P- value < 0.0001 KW 51.585

1, 2 and 3 was 100%, 40% and 40% respectively. Moreover, mean of antibody in smokers of three groups was 38.02-± 10, 12.7± 7.6 and 4.2 ± 1.3 U/ ml respectively. Using Dunn compare test, the mean difference of antibody in groups 1 and 3 was found to be statistically significant (p<0.05) but differences between groups 1 and 2 and groups 2 and 3 were not significant (p> 0.05)

Discussion

Our study showed a high frequency of serum positivity for anti-CP antibody more than 5 units in ml) in patients with coronary artery disease especially patients suffering from acute myocardial infarction. Marinella and his collegues reported that levels of anti-CP and anti-cytomegalvirus antibody in patients with acute MI were higher than that amongst control- group9. These results corresponded to our study. In this study the risk of CP infection in smokers was more than others. In our study also mean concentration and Frequency of serum positivity of anti-CP antibody in smokers were more than non-smokers. These results confi rm that smoking increases susceptibility to CP infection.

In a study done by Gupta et al, 213 male patients with previous MI were studied and results showed that in 90% of patients, amount of anti-CP IgG antibody had increased and remained instant within 3 months7. This result corresponds to our results which in all patients with MI, the level of antibody was more than 5 unit in ml. Higher percentage obtained in our study maybe was resulting from reciprocal reaction of kite with heart shock protein entered to serum while occurring of MI. Moreover, they reported 11% of controls had positive anti-CP 1gG antibody, but in our study it was 31% which can be attributed to the higher number of smokers in our study. In Gupta’s study, patients with MI were studied during the last 6 months, while in our study patients with new MI were studied (first week after MI).

Ridker et al first measured anti-CP antibody in 1500 healthy men (aged 40-80 years) and followed them for a period of 12 years. They found that there was no evidence showing the relationship between positive anti-CP antibody and risk increase of MI in future. In this study, the incidence of positive anti-CP antibody was 68% and there was a direct association with age and smoking3, But in our study, incidence of positive anti-CP antibody in control group was 31%. This difference can be related to lower mean age and lower number of smoker in our study.

Withene and his colleagues performed a study on 121 patients with first attack of MI and 204 control subject with the same age, sex, race. They found that there was no association between CP infection and Ml.16 The results of this study has no corresponding to Our study because in our study patients were selected in acute phase of MI resulting in higher prevalence of positive anti-CP antibody.17

With comparing the results of above studies and results of obtained in our study, the role of chlamydia infections and IgG anti- CP antibody in patients with coronary heart diseases especially in acute MI is corroborated. Since there is evidence from previous studies 18 of importance of IgG as compared to other antibodies (CPIgA/IGM) and also measurement of IgG in studies on effect of 19,7 antibiotics we did not try to measure there. It has recently been reported that in autopsies done on human coronary arteries, there will a direct correlation between titrations of IgG (not IgA or IgM) and determining chlamydia infection.

The result, of our study showed that patients with Coronary Artery Disease especially patients with acute MI had higher mean and frequency of positive anti- CP antibody as compared to that in control group. Therefore, the results suggest that probably there is a relationship between chlamydia infection and coronary artery diseases (CAD) specially acute MI.

Limitation

One of the limitations in our study was measuring antibody in which there was the probability of reciprocal reaction with other microorganisms, especially different kinds of chlamydia. By using Eliza method and a completely specifi c kite against CP antigen, we minimized this probability.

Acknowledgement and Appreciation

This plan was done by using the research credits of Rafsanjan University of medical sciences. We sincerely appreciate from related authors and also Ms. Alipoor for typing the article.

References

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Deptt. of Biostatistics and Epidemiology, Health Faculty affiliated to Shahid Sadoghi. University of Medical Sciences and Health Services, Daneshjue Blv. Yazd, Islamic Republic Of Iran.
E-mail: m_h_lotfi @yahoo.com
Received: 25.6.04

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