Prevalence of Helicobacter pylori, cytomegalovirus, and Chlamydia pneumoniae immunoglobulin seropositivity in coronary artery disease patients and normal individuals in North Indian population

Abstract

Background

In present day atherosclerosis is perceived as a chronic inflammatory vascular condition and infectious diseases are believed to contribute to its pathophysiology. In this context, the microorganisms which are believed to play a role in the pathophysiology include Chlamydia pneumoniae, cytomegalovirus (CMV), and Helicobacter pylori.

Method

A case control study (retrospective) was conducted over a two-year period. The study population was divided into two groups with 200 individuals in each group. The first group comprised cases of coronary artery disease (CAD) and the second comprised healthy controls selected from the general population after matching for age and sex. Enzyme-linked immunosorbent assay (ELISA) was done for immunoglobulin (IgG) antibodies to H. pylori, C. pneumonia, and CMV. They were also evaluated for conventional risk factors including hypertension, diabetes, obesity, and dyslipidaemia. Epi Info™ version 6 six software was used for analysis of data. Odds ratio, χ² for trend and multiple logistic regression analysis were used to find out statistically significant results.

Results

Seropositivity for H. pylori was present in 119 patients of CAD (59.5%) but it was present in only 76 controls (38%) (P = 0.001). There was a statistically significant association between seropositivity for H. pylori and CAD. There was no statistically significant association between C. pneumoniae and CMV seropositivity with CAD. Multiple logistic regression analysis was done with CAD as the outcome (dependent variable). The predictor covariates (independent) variables were seropositivity to H. pylori, C. pneumoniae, and CMV, hypertension, obesity, diabetes, and dyslipidaemia. It was found that seropositivity to H. pylori, hypertension, obesity, and dyslipidaemia were significant risk factors for CAD.

Conclusion

Our study shows an association between IgG antibody response to H. pylori and CAD. Multiple logistic regression analysis showed that this association was retained even on comparison with the other risk factors.

Introduction

Atherosclerosis is the main underlying cause of cardiovascular diseases. Framingham's heart study and other studies have shown main risk factors of coronary artery disease (CAD) including hypertension, hypercholesterolaemia, diabetes, smoking, environmental factors (such as unsuitable diets, less physical activity), and hereditary factors.^1,2 One of the first published texts related to the atherogenic role of virus, namely, Marek's disease herpervirus, appeared in 1978.³ At present, several infectious agents resulting in chronic infections have been specified as probable contributors in generation of atherosclerosis.⁴ Microorganisms believed to play a role include Chlamydia pneumoniae, cytomegalovirus (CMV), and Helicobacter pylori.

The conventional atherosclerosis risk factors, including hypertension, diabetes mellitus (DM), smoking, and cardiac diseases, do not fully account for the risk of atherosclerosis, and cases, especially young subjects, often do not have any of these factors.⁵ Atherosclerosis is today perceived as a chronic inflammatory vascular condition.⁶

Materials and Method

A case control study (retrospective) was conducted. The study population was divided into two groups. The first group comprised the cases of CAD selected on a combined account of clinical history, electrocardiographic (ECG) changes, and treatment records. The second group comprised healthy controls selected from the general population after matching for age and sex. Both groups together comprised 200 individuals. The study was conducted over a period of two years at a zonal hospital in the Northern sector.

The subjects were interviewed in the outpatient department (OPD) of the hospital. Before the interview the subjects were informed about the scope and nature of the study and were fully assured strict confidentiality. Factual data was recorded by the investigator. For physical examination and blood sample collection for biochemical and serological testing, subjects were called to the hospital laboratory and at that time the data was cross checked and corroborated with the available records with the patient.

For purpose of data collection, age was defined in years. It was ascertained by dependent card/service identity card. History of hypertension was asked for and the patient's notebook containing treatment details was seen for confirmation of diagnosis. Three successive readings were taken at 5 minute intervals and the average was recorded as the blood pressure. The scale used for detection was according to the Seventh Report of the Joint National Committee on prevention, detection, evaluation, and treatment of high blood pressure.⁷

A cut-off fasting plasma glucose level of ≥ 126 mg% was taken as diabetic. The subject was also considered diabetic if he/she was already a known diabetic under treatment.

World Health Organization (WHO) classification was used to define obesity. Height of the subject measured in nearest cm and weight in Kg was recorded. Body mass index (BMI) was ascertained and values above 25 were considered as obese.⁸

The subjects were stratified based on lipid values as per the adult treatment panel (ATP) III classification according to Third Report of the National Cholesterol Education Programme (NCEP) expert panel on detection, evaluation, and treatment of high blood cholesterol in adults.⁹

Fasting blood samples were collected for testing after consent. Kits used for biochemical testing (plasma glucose and serum lipid profile) were manufactured by Transasia Biomedicals Ltd in technical collaboration with Erba Diagnostics, Germany. For enzyme-linked immunosorbent assay (ELISA), serum from blood samples of control and study groups were stored at −20°C until analysis. All serum samples were evaluated for presence of IgG antibodies to H. pylori by ELISA using kits manufactured by Biotecx Laboratories, Houston, Texas. The manufacturers claimed a sensitivity and specificity of 100%. Immunoglobulin G antibodies to CMV was tested by ELISA (kits manufactured by Biotecx Laboratories, Houston, Texas) and the manufacturers claimed a sensitivity and specificity of 100%. Immunoglobulin G antibodies to C. pneumoniae were detected by ELISA using kits manufactured by Novatech Immunodiagnostica, Germany.

Data was compiled on an excel sheet. Epi Info™ version 6 software was used for analysis of data. Odds ratio, χ² for trend, and multiple logistic regression analysis were used for statistical analysis.

Results

The median age for cases and controls was 51 was and 54 years, respectively. Around 72% of the cases and 75% of the controls were in the age group of 46–85 years (Table 1); 53% of cases and 46% of controls were males.

Table 1.

Distribution of cases and controls with respect to age.

Age group (yr)	Cases (% of total number)	Controls (% of total number)
	n = 200	n = 200
30–45	52 (26)	48 (24)
46–60	86 (43)	90 (45)
61–85	58 (29)	60 (30)
> 85	4 (2)	2 (1)

Immunoglobulin G (IgG) seropositivity for H. pylori was present in 119 patients (59.5%) but it was present in only 76 controls (38%) (P = 0.001; Table 2). There was a statistically significant association between seropositivity for H. pylori and CAD. Odds that an individual with CAD had H. pylori seropositivity were 2.40 times greater than for an individual without CAD. The prevalence of H. pylori seropositivity was seen to increase with increasing age of the subjects with CAD and this relation was statistically significant. The prevalence of H. pylori seropositivity in controls decreased initially from the age group up 30–45 (33.33%) to 46–60 (22.20%), however it increased in the group of 61–87 years of age (64.51%) and no linear trend was seen.

Table 2.

Distribution of cases and controls with respect to Helicobacter pylori infection.

	Seropositive for H. pylori	Seronegative for H. pylori
Cases of CAD (n = 200)	119	81
Controls (n = 200)	76	124
Proportion of cases exposed: 0.595
Proportion of controls exposed: 0.38

CAD: coronary artery disease, H. pylori: Helicobacter pylori.

χ² value 18.5

P value (0.000); < 0.05

Odds ratio 2.40 (95% CI: 1.57–3.66)

Seropositivity for H. pylori was present in 119 patients (59%) but it was present in only 76 controls (38%) (P = 0.001).

Immunoglobulin G seropositivity for C. pneumoniae was present in 130 patients (65%) and in 128 controls (64%). There was no statistically significant association between the presence of Chlamydia infection and CAD. The prevalence C. pneumoniae seropositivity was seen to increase with increasing age of the subjects from age group of 30–45 years (64.58%) to 46–60 years (73.33%); however, it declined in the age group of 61–85 years (50%). There was no linear trend observed between increasing age and chlamydia seropositivity.

Immunoglobulin G seropositivity for CMV was present in 122 patients (61%) while it was present in 130 controls (65%). There was no statistically significant association between the presence of CMV infection and CAD. The prevalence of CMV seropositivity in controls was static, about 75%, in the age groups 30–45 and 46–60 years. Seropositivity to CMV infection declined in controls in the age group 60–87 years (40.6%).

Multiple logistic regression analysis was done with CAD as the outcome (dependent variable; Table 3). The predictor covariates (independent) variables were IgG seropositivity to H. pylori, C. pneumoniae, and CMV, hypertension, obesity, diabetes, and dyslipidaemia. It was found that IgG seropositivity to H. pylori, hypertension, obesity, and dyslipidaemia were significant risk factors for CAD.

Table 3.

Results of multiple logistic regression analysis of various risk factors for association with coronary artery disease.

Term	Odds ratio	95% CI		Coefficient	SE	Z statistic	P value
C. pneumoniae	1.9241	0.9032	4.0990	0.6545	0.3859	1.6961	0.0899
CMV	1.7332	0.7756	3.8732	0.5500	0.4103	1.3405	0.1801
H. pylori	2.1978	1.0758	4.4900	0.7874	0.3645	2.1603	0.0307
DM	0.6799	0.3325	1.3903	−0.3858	0.3650	−1.0572	0.2904
Hypertension	2.9977	1.4496	6.1992	1.0979	0.3707	2.9616	0.0031
Lipid profile	8.2611	3.8953	17.5201	2.1116	0.3836	5.5049	0.0000
Obesity	3.2546	1.5560	6.8076	1.1801	0.3765	3.1342	0.0017

CMV: cytomegalovirus, C. pneumoniae: Chlamydophila pneumoniae, DM: diabetes mellitus, H. pylori: Helicobacter pylori, SE: standard error.

Multiple logistic regression analysis was done with coronary artery disease (CAD) as the outcome (dependent variable). The predictor covariates (independent variables) were C. pneumoniae, CMV, H. pylori, DM, hypertension, dyslipidaemia, and obesity. It was found that H. pylori, hypertension, obesity, and dyslipidaemia were significant risk factors for CAD.

Discussion

Infection with H. pylori is common in the Indian subcontinent. Exposure occurs in childhood and approximately 80% of adults have been infected at some time. Prasad et al had demonstrated a seroprevalence of 80% in asymptomatic healthy individuals in the community.¹⁰

Seropositivity for IgG antibodies to H. pylori was seen in 59.5% of the cases as against 38% of the controls. A significant association was present between presence of H. pylori infection and CAD. This is similar to the findings of Mendall et al¹¹ and Patel et al.¹² This suggests that H. pylori may be involved in the pathogenesis of atherosclerosis. A study has confirmed identification of H. pylori DNA in atherosclerotic plaques of patients with severe CAD. This supports the hypothesis that H. pylori may be involved in the pathogenesis of atherosclerosis.¹³ Another study suggested that in younger individuals in Japan, H. pylori infection is significantly associated with acute myocardial infarction (AMI) independent of the classic coronary risk factors.¹⁴ This was also seen in our study where H. pylori seropositivity proved to be an independent risk factor for CAD on multiple logistic regression analysis independent of other conventional risk factors. Rajasekhar et al had shown higher levels of lipids, lipoproteins, C-reactive protein and higher percentage of coronary risk factors in patients seropositive to H. pylori in unstable angina suggesting that H. pylori may influence coronary risk factors and may play a role in pathogenesis of atherosclerosis.¹⁵ Infection with H. pylori may influence atherogenesis through low grade, persistent inflammatory stimulation.¹⁶ Other mechanisms for the development of atherosclerosis in relation to H. pylori infection have also been studied. Tamura et al from Japan suggested that H. pylori-induced chronic atrophic gastritis decreases plasma vitamin B₁₂ and folic acid levels, thereby increasing homocysteine levels, which is a known risk factor for atherosclerosis.¹⁷

Investigators looked at serological markers of infection with H. pylori in a group of patients with unstable angina. However, no relationship was found between unstable angina and H. pylori infection, though relationship with C. pneumoniae and CMV was demonstrable.¹⁸ Eradication of H. pylori infection does not appear to influence any changes of coronary risk factors, as reported by one study that compared sugar, lipid, and fibrinolytic profiles before and after H. pylori eradication.¹⁹ It has been shown that no benefit of treatment with antibiotics was observed in high-risk patients as reported by trials examining whether treatment of infection can prevent the complications of CAD.²⁰

Sero-surveys indicate a seroprevalence of 22–57% in children under the age of five, increasing to 80–90% by the age of 20, and remaining constant thereafter.^21–23 The prevalence in our study was lower than that demonstrated in these studies. This may be due to the fact that the prevalence of H. pylori varies with geographic area, ethnicity, race, age, and socioeconomic status.^21,22

Immunoglobulin G antibody seropositivity for C. pneumoniae was present in 130 patients (65%) and in 128 controls (64%). There was no significant association between presence of Chlamydia infection as evidenced by presence of seropositivity to IgG antibodies and CAD. This finding is similar to the finding of Sotiropoulos et al.²⁴ In several epidemiological and experimental studies, C. pneumoniae, among other infectious agents, has been associated with the risk of ischaemic heart disease.^25,26 Serological association was also shown by Agarwal et al.²⁷ They showed a seroprevalence of 64% in the cases (similar to ours) but a lower seroprevalence of 38% in the control group. The prevalence of C. pneumoniae seropositivity was seen to increase with increasing age of the subjects from age group of 30–45 years (64.58%) to 46–60 years (73.33%); however, it declined in the age group of 61–85 years (50%). There was no linear trend observed between increasing age and seropositivity for IgG antibodies to C. pneumoniae. A study by Koh et al demonstrated C. pneumoniae seropositivity of 74.6% in males and 59.4% in females.²⁸ They also demonstrated rising seropositivity with increasing age from childhood to adulthood, with subsequently stable levels. This pattern of rising and levelling off of seropositivity with age suggests that C. pneumoniae infection occurs early in life, and in older ages the high level of seropositivity is probably maintained by reinfections or chronic infections.

Seropositivity for CMV was present in 122 patients (61%) and it was present in 130 controls (65%). There was no significant association between presence of CMV infection and CAD.

Our study demonstrated a prevalence of CMV infection as evidenced by elevated IgG antibodies of 65% in the control group. The prevalence of CMV seropositivity in controls was static, about 75%, in the age groups 30–45 and 46–60 years. Seropositivity to CMV infection declined in controls in the age group 60–87 years.

The prevalence of IgG antibodies to CMV in Indian population has earlier been shown to be around 95%.²⁹ These studies did not demonstrate any significant difference of seropositivity to CMV based on age. This differs from Western studies which show a significantly increased seropositivity with age.³⁰ This may be due to the early acquisition of the infection in India in childhood compared to Western populations leading to a higher prevalence in younger adults.

We were able to demonstrate association of CAD with dyslipidaemia, hypertension, and obesity. However, we were not able to demonstrate any association with DM. The independent risk on logistic regression analysis was found to be highest for dyslipidaemia.

Conclusion

Our study showed a statistically significant association between seropositivity for IgG antibodies to H. pylori and CAD. Associations of CAD with dyslipidaemia, hypertension, and obesity were also demonstrable.

On the basis of current knowledge, chronic infections like H. pylori appear to be risk factors that may act in association with conventional risk factors and genetic predispositions and are not sufficient for disease development. In such a concept, Koch's postulates for causality, which require a specific link between the microbial agent and the disease, among others, cannot be satisfied. However, more general criteria for causality are still insufficiently fulfilled, and the role of chronic infection and inflammation in atherosclerosis pathogenesis is still incompletely defined. Future studies need to address whether inhibition of inflammation or longstanding antimicrobial therapies will reduce the risk of atherosclerosis and CAD and thus offer effective adjuncts to treatment already in clinical use.

Intellectual Contributions of Authors

Study concept: Lt Col Rohit Tewari

Drafting and manuscript revision: Lt Col Rohit Tewari, Col MN Mishra, Lt Col Puja Dudeja

Statistical analysis: Lt Col Puja Dudeja

Study supervision: Brig TK Salopal, Col VS Nijhawan

Conflicts of Interest

None identified.