Seroprevalence of Antibodies to Microorganisms Known To Cause Arterial and Myocardial Damage in Patients with or without Coronary Stenosis

Abstract

Infections are assumed to play a role in coronary artery disease (CAD) and cardiomyopathies. It is unknown whether the seroprevalence of antibodies to these microorganisms is higher in patients with than without CAD. The seroprevalence of antibodies to Bartonella henselae, Borrelia burgdorferi, Chlamydia pneumoniae, Coxiella burnetii, Helicobacter pylori, human granulocytic Ehrlichia, Leptospira, Rickettsia conorii, and Treponema pallidum was assessed prospectively in patients with exertional dyspnea or anginal chest pain who underwent coronary angiography because of suspected CAD. Patients with normal angiograms (NA) were those in whom no more than 50% stenosis of any coronary artery was found. Patients with CAD were patients who underwent percutaneous transluminal coronary angioplasty. There were 50 patients with CAD (9 female) and 62 with NA (25 female), with a mean age of 62 years. All patients had antibodies to at least one microorganism: to B. henselae, 8% of CAD patients and 5% of NA patients; to B. burgdorferi IgG, 14% CAD and 6% NA; to B. burgdorferi IgM, 6% CAD and 3% NA; to C. pneumoniae lipopolysaccharide (LPS) IgA, 76% CAD and 77% NA; to C. pneumoniae LPS IgG, 80% CAD and 90% NA; to C. burnetii, 0% CAD and 5% NA; to H. pylori, 92% CAD and 68% NA; to human granulocytic Ehrlichia, 8% CAD and 3% NA; to Leptospira IgG, 4% CAD and 2% NA; to R. conorii, 10% in both groups; and to T. pallidum, 2% CAD and 0% NA. The seroprevalence of antibodies to micro-organisms known to induce arterial and myocardial damage does not differ between patients with CAD and NA.

Infections are assumed to play a role in the pathogenesis of coronary artery disease (CAD) and cardiomyopathies. Among microorganisms known to cause arterial and myocardial damage are Bartonella henselae, Borrelia burgdorferi, Chlamydia pneumoniae, Coxiella burnetii, Helicobacter pylori, human granulocytic Ehrlichia, Leptospira, Rickettsia conorii, and Treponema pallidum (4, 8, 13, 14, 16, 26, 27, 28). It is unknown, however, whether the seroprevalence of antibodies to these microorganisms is higher in patients with coronary heart disease than in those without coronary heart disease who suffer from exertional dyspnea or anginal chest pain. Therefore, a prospective study was carried out with patients with exertional dyspnea or anginal chest pain who underwent a coronary angiography because of suspected coronary heart disease based on pathologic findings on stress test, scintigraphy, and echocardiography. Aims of the study were (i) to compare the seroprevalence of specific antibodies to microorganisms known to cause arterial and myocardial damage between patients with normal angiograms and patients with coronary heart disease and (ii) to assess the seroprevalence of antibodies to microorganisms known to cause arterial and myocardial damage in patients with normal angiograms with regard to (a) possible causes of exertional dyspnoea and anginal chest pain, like arterial hypertension, hemochromatosis, hypothyroidism, hypoparathyroidism, tachycardiomyopathy, amyloidosis, and neuromuscular disorders, and (b) echocardiographic findings (17).

MATERIALS AND METHODS

Patients.

The group of patients with normal angiograms consisted of consecutive patients in whom a coronary angiography had been performed because coronary heart disease was suspected by clinical findings (exertional dyspnea or anginal chest pain) and noninvasive tests (stress test, scintigraphy, and echocardiography) and no relevant (>50%) stenosis of any coronary artery had been found. Excluded were patients in whom a coronary angiography was performed prior to valve surgery or organ transplantation. The group of patients with coronary heart disease consisted of consecutive patients who underwent percutaneous transluminal coronary angioplasty (PTCA) of one or more coronary artery stenosis. The coronary angiographies and PTCA were performed at the 2nd Medical Department of the Krankenanstalt Rudolfstiftung. All patients of both groups were invited for a follow-up visit between April and July 1999. During this visit, patients with normal coronary angiograms underwent extensive investigations, including a medical history, physical examination, 12-lead electrocardiogram (ECG), transthoracic 2-D, M-mode and Doppler echocardiography, and blood tests (blood sedimentation rate, red and white blood cell counts, thrombocyte count, transferrin saturation, creatine kinase, γ-glutamyl-transpeptidase, calcium, potassium, thyroid stimulating hormone, and analysis of the hemochromatosis gene mutations C282Y and H63D). Based on these investigations, possible causes of dyspnea and anginal chest pain were assessed according to predefined criteria (Table 1) (17). Patients with coronary heart disease underwent clinical examination and exercise testing 3 months after PTCA. At this visit, blood was taken from both groups for serologic investigations comprising specific antibodies to the following microorganisms, which are known to cause arterial and myocardial damage: B. henselae, B. burgdorferi, C. pneumoniae, C. burnetii, H. pylori, human granulocytic Ehrlichia, Leptospira, R. conorii, and T. pallidum. The test systems used are listed on Table 2. All tests were performed according to the manufacturer's instructions at one institute (Klinisches Institut für Hygiene der Universität Wien). Informed consent was obtained from all patients, and the study was approved by the institutional ethics committee.

TABLE 1.

Diagnostic criteria for possible causes of symptoms and their prevalence in group of patients with normal angiograms

Cause of symptoms	Diagnostic criteria	Prevalence (n)
Arterial hypertension	History of arterial hypertension or treatment with antihypertensive drugs	44
Haemochromatosis	Homozygous for HFE gene mutation (Cys 282→Tyr) or HFE gene mutation (His 63→Asp) or heterozygous for both mutations (7, 19)	2
Hypothyroidism	Elevated TSH values (>4.0 μIE/ml)	3
Hypoparathyroidism	Decreased serum Ca (<2.1 mmol/liter) and parathormone values (12)	0
Tachycardiomyopathy	Heart rate of >100/min at resting ECG and decreased systolic function (2)	5
Amyloidosis	Biopsy of the involved tissue, showing amyloid deposition (21)	0
Neuromuscular disorder	Evidence from medical history, clinical examination, biochemical data, and electromyogram of a myopathy or polyneuropathy (9)	7
Unknown	Absence of any of the above-mentioned abnormalities	14

TABLE 2.

Test systems used in order to identify specific antibodies to selected pathogens in sera of patients with cardiac symptoms^a

Pathogen	Test system	Antigen	Immunoglobulin class(es) determined	Reading photometer wave length	Positive result criterion	Manufacturer
Bartonella henselae	Immunofluorescent assay	Infected Vero cells; coated on slides	IgG	Fluorescence microscope, 470- to 490-nm filter	Comparison with positive and negative controls	MRL Diagnostics
Borrelia burgdorferi	EIA	Microwells coated with B. garinii (strain W) whole-cell supernatant sonicate	IgG, IgM	450 nm	OD ≥ 0.350	In house
Chlamydia pneumoniae	Immunofluorescent assay	Purified elementary bodies fixed on slides	IgA, IgG	Fluorescence microscope, 470- to 490-nm filter	Comparison with positive and negative controls	MRL Diagnostics,
Coxiella burnetii	EIA	Microwells coated with purified whole cells cultured in embryonated eggs	IgG	405 nm	≥30 U/ml	Virion-Serion, Würzburg, Germany
Helicobacter pylori	EIA	Microwells coated with H. pylori whole-cell supernatant sonicate	IgG	450 nm	OD ≥ 0.120	Meridian Diagnostics, Milan, Italy
Human granulocytic Ehrlichia	Immunofluorescent assay	Inactivated HGE cells fixed on slides	IgG	Fluorescence microscope, 470- to 490-nm filter	Comparison with positive and negative controls	MRL Diagnostics,
Leptospira	EIA	Microwells coated with L. biflexa purified whole-cell sonicate	IgG	405 nm	≥ 9 U/ml	Virion-Serion, Würzburg, Germany
Rickettsia conorii	Immunofluorescent assay	Inactivated R. conorii cells fixed on slides	IgG	Fluorescence microscope, 470- to 490-nm filter	Comparison with positive and negative controls	MRL Diagnostics,
Treponema pallidum	EIA	Microwells coated with T. pallidum cells	IgG	450 nm	≥2× negative control	Gull Laboratories Bad Homburg, Germany

^aEIA, enzyme immunoassay; OD, optical density. 

RESULTS

Included in the study were 112 patients, 50 with coronary heart disease who underwent PTCA between January and April 1999 and 62 with normal coronary angiograms performed between January and December 1998. The group with coronary heart disease consisted of 9 female and 41 male patients with a mean age of 62 years. In the group with normal coronary angiograms (25 female, 37 male, mean age of 62 years), the patients suffered from anginal chest pain (n = 38 [61%]), exertional dyspnea (n = 12 [19%]), or a combination of exertional dyspnea and anginal chest pain (n = 12 [19%]). In this group, all patients except one underwent one or more noninvasive tests prior to coronary angiography: echocardiography (n = 25), bicycle stress test (n = 3), echocardiography and bicycle stress test (n = 14), echocardiography and scintigraphy (n = 6), bicycle test and scintigraphy (n = 1), or echocardiography, bicycle test, and scintigraphy (n = 12). In patients with normal coronary angiograms, 47 patients had smooth normal vessels and 15 patients had coronary sclerosis but no significant (>50%) stenosis. In 48 of the 62 patients with normal coronary angiograms, one or more causes of cardiac symptoms could be assessed: arterial hypertension (n = 44), hemochromatosis (n = 2), hypothyroidism (n = 3), tachycardiomyopathy (n = 5), and neuromuscular disorder (n = 7). In the remaining 14 patients with normal coronary angiograms, the cause of exertional dyspnea and anginal chest pain remained unknown (Table 1) (17). The seroprevalence of antibodies known to cause arterial and myocardial damage is listed on Table 3. All included patients had antibodies to at least one microorganism. The seroprevalence was similarly distributed between patients with coronary artery stenosis and with normal coronary angiograms. Antibodies to C. pneumoniae and H. pylori were most prevalent in both groups.

TABLE 3.

Seroprevalence of antibodies to microorganisms known to cause arterial and myocardial damage in patients with exertional dyspnea with coronary heart disease and with normal coronary angiograms^a

Microorganism	No. (%) of patients with specific antibodies to microorganism
	Coronary heart disease	Normal coronary angiogram
Bartonella henselae	4 (8)	3 (5)
Borrelia burgdorferi IgG	7 (14)	4 (6)
Borrelia burgdorferi IgM	3 (6)	2 (3)
Chlamydia pneumoniae LPS IgA	38 (76)	48 (77)
Chlamydia pneumoniae LPS IgG	40 (80)	56 (90)
Coxiella burnetii	0 (0)	3 (5)
Helicobacter pylori	46 (92)	42 (68)
Human granulocytic Ehrlichia	4 (8)	2 (3)
Leptospira IgG	2 (4)	1 (2)
Rickettsia conorii	5 (10)	6 (10)
Treponema pallidum	1 (2)	0 (0)
Total	50	62

^aLPS, lipopolysaccharide. 

In the group with coronary heart disease, five patients had antibodies only to H. pylori, and four patients had only C. pneumoniae IgG and IgA. Antibodies to two different microorganisms were found in 25 patients, most commonly a combination of antibodies to H. pylori and C. pneumoniae IgG and IgA (n = 19). Antibodies to three microorganisms were found in nine patients, to four microorganisms in five patients, and to five microorganisms in two patients.

In the group with normal coronary angiograms, 3 patients had antibodies only to H. pylori and 14 patients had antibodies only to C. pneumoniae IgG and IgA. Antibodies to two different microorganisms were found in 35 patients, most commonly a combination of antibodies to H. pylori and to C. pneumoniae IgG and IgA (n = 21). Antibodies to three microorganisms were found in seven patients and to four microorganisms in three patients. The seroprevalence of antibodies in the group with normal coronary angiograms with regard to the possible causes of their symptoms and echocardiographic findings are listed on Tables 4 and 5. Within the group with normal coronary angiograms, the seroprevalence did not differ between patients with smooth normal vessels and patients with coronary sclerosis (data not shown).

TABLE 4.

Antibodies to microorganisms known to cause arterial and myocardial damage, possible causes for cardiac symptoms, and echocardiographic findings in 48 patients with normal coronary angiograms^a

Target microorganism(s) of antibody	Patient characteristics
	Age/sex	Cause(s)	Echocardiogram result(s)
Helicobacter pylori and no other antibodies	36/M	AH	LVH
	74/M	AH	DIL, SYD, LVH
	52/M	AH, TM	DIL, LVH, DIA
Helicobacter pylori, Chlamydia pneumoniae LPS IgG	72/M	AH	Normal
	68/F	AH	LVH, DIA, ILVAT
	60/F	AH	LVH
	62/M	AH	DIL, LVH
	68/F	AH	DIL, LVH, DIA
	68/F	AH, HY	LVH, DIA
Helicobacter pylori, Chlamydia pneumoniae LPS IgG, Chlamydia pneumoniae LPS IgA	59/M	HY	LVH, SYD
	72/F	HC	DIL, LVH, SYD
	62/F	AH	Normal
	57/M	AH	LVH, DIA
	69/M	AH	Normal
	72/F	AH	Normal
	78/F	AH	LVH
	47/M	AH	LVH, SYD
	54/F	AH	LVH, ILVAT
	69/F	AH	LVH, DIA
	60/M	AH	LVH, SYD
	79/M	AH	DIL, LVH, SYD
	57/M	AH	LVH
	48/M	AH, NM	DIL, LVH, SYD, ILVAT
	55/M	AH, TM	DIL, LVH, SYD
	51/M	AH, TM	LVH, DIA
Helicobacter pylori Human granulocytic Ehrlichia, Chlamydia pneumoniae LPS IgG, Chlamydia pneumoniae LPS IgA	40/F	AH	LVH
	65/F	AH	LVH, DIA
Helicobacter pylori, Borrelia burgdorferi IgG, Rickettsia conorii, Chlamydia pneumoniae LPS IgG, Chlamydia pneumoniae LPS IgA	61/M	AH	DIL, LVH, SYD
Helicobacter pylori, Coxiella burnetti, Chlamydia pneumoniae LPS IgG	59/M	AH	LVH
Helicobacter pylori, Coxiella burnetti, Chlamydia pneumoniae LPS IgG, Chlamydia pneumoniae LPS IgA	53/M	AH	LVH, SYD
Helicobacter pylori, Bartonella henselae, Rickettsia conorii	54/M	AH	LVH, DIA
Chlamydia pneumoniae LPS IgG	74/F	AH	LVH, DIA
Chlamydia pneumoniae LPS IgG, Chlamydia pneumoniae LPS IgA	55/M	NM	DIL, SYD, ILVAT
	73/F	AH	LVH, DIA
	64/F	AH	LVH, SYD, ILVAT
	59/F	AH	Normal
	64/M	AH	LVH
	74/M	AH	DIL, LVH, DIA
	70/F	AH, HC	DIA
	54/M	AH, NM	DIL, LVH, DIA, ILVAT
	58/M	AH, NM	LVH, ILVAT
	30/M	AH, NM	LVH
	45/M	AH, TM	LVH, DIA
	52/M	AH, NM, TM	DIL, SYD, ILVAT
Rickettsia conorii, Chlamydia pneumoniae LPS IgG, Chlamydia pneumoniae LPS IgA	64/F	AH	LVH, DIA
	59/M	AH, NM	DIL, LVH, SYD, ILVAT
Bartonella henselae, Chlamydia pneumoniae LPS IgG, Chlamydia pneumoniae LPS IgA	56/F	AH, HY	Normal
Leptospira IgG, Chlamydia pneumoniae LPS IgG	69/M	AH	LVH, SYD

^aAH, arterial hypertension; DIA, diastolic dysfunction (E/A ratio of <1 or restrictive filling pattern); DIL, left ventricular enddiastolic diameter of >57 mm; HC, hemochromatosis; HY, hypothyroidism; ILVAT, isolated left ventricular abnormal trabeculation; LVH, left ventricular wall thickness of >11 mm; NM, neuromuscular disorder; SYD, left ventricular fractional shortening of <30%; TM, tachycardiomyopathy. 

TABLE 5.

Antibodies to microorganisms known to cause arterial and myocardial damage and echocardiographic findings for 14 patients with normal coronary angiograms with no possible causes for cardiac symptoms

Target microorganisms of antibody	Patient characteristics
	Age/sex	Echocardiogram result(s)a
Helicobacter pylori, Chlamydia pneumoniae LPS IgA	49/F	Normal
Helicobacter pylori, Chlamydia pneumoniae LPS IgG, Chlamydia pneumoniae LPS IgA	49/M	LVH, DIA
	60/M	LVH
	58/M	DIL, LVH, SYD
	62/F	DIL, LVH, SYD
	75/F	LVH, DIA
Helicobacter pylori, Chlamydia pneumoniae LPS IgG, Chlamydia pneumoniae LPS IgA, Borrelia burgdorferi IgM	39/F 51/M	Normal LVH, DIA, ILVAT
Helicobacter pylori, Borrelia burgdorferi IgG, Rickettsia conorii, Chlamydia pneumoniae LPS IgG, Chlamydia pneumoniae LPS IgA	76/M 69/M	DIL, LVH, SYD DIL, SYD
Helicobacter pylori, Bartonella henselae	43/F	Normal
Chlamydia pneumoniae LPS IgG, Chlamydia pneumoniae LPS IgA	61/M	LVH
Borrelia burgdorferi IgG, Chlamydia pneumoniae LPS IgG, Chlamydia pneumoniae LPS IgA	71/M	DIL, LVH
Coxiella burnetii, Chlamydia pneumoniae LPS IgG, Chlamydia pneumoniae LPS IgA	37/F	Normal

^aDIA, diastolic dysfunction (E/A ratio of <1 or restrictive filling pattern); DIL, left ventricular enddiastolic diameter of >57 mm; ILVAT, isolated left ventricular abnormal trabeculation; LVH, left ventricular wall thickness of >11 mm; SYD, left ventricular fractional shortening of <30%. 

DISCUSSION

This study shows that the seroprevalence of microorganisms known to induce arterial and myocardial damage is similar in patients with exertional dyspnea and anginal chest pain, irrespective if they have normal coronary angiograms or coronary heart disease. In patients with normal angiograms, the seroprevalence of antibodies to microorganisms known to cause arterial and myocardial damage does not differ with regard to (i) possible causes of exertional dyspnea and anginal chest pain, like arterial hypertension, hemochromatosis, hypothyroidism, tachycardiomyopathy, and neuromuscular disorders, and (ii) echocardiographic findings.

An association of coronary heart disease and infections with C. pneumoniae, an important respiratory pathogen, has been initially found by seroepidemiologic studies (23). More recently, the presence of C. pneumoniae in atheromatous plaques has been shown (3). At present, controversial results are available about the role of C. pneumoniae in the development of coronary heart disease. Several authors found an association (18, 24) whereas others did not (5, 6). Another microorganism suspected to play a role in coronary artherosclerosis is H. pylori, the main etiological factor in gastritis and peptic ulcer disease. H. pylori infection is postulated to have an effect on clotting mechanisms and lead to a prothrombotic state (10). Again, controversial results are available about the role of H. pylori in the development of coronary heart disease. Several authors found an association (20), whereas others did not (5, 10, 18), or found an association which can be adequately explained by the much stronger association of H. pylori infection with age and social class, both of which are linked with coronary heart disease (15). Nearly all our patients with coronary stenosis (92%) had antibodies to H. pylori, whereas in the group with normal coronary angiograms, they were found in only 68% of the patients. This finding may be due to the differing proportions of male patients in the group with coronary heart disease (82%) and with normal coronary angiograms (60%). Furthermore, epidemiological studies show that the seroprevalence of antibodies to H. pylori is generally higher in men than women (15, 22). One reason for the controversial results about the association of infections with C. pneumoniae or H. pylori with coronary heart disease might be that some of the studies compared patients with coronary heart disease and asymptomatic controls (6, 18, 20, 23, 24), whereas other studies, like the present one, compared symptomatic patients with and without coronary artery stenosis (10).

In the absence of coronary heart disease, exertional dyspnea and anginal chest pain may be caused by extracardiac causes, like pulmonary, skeletal, gastrointestinal, haematological, or neurologic disorders, by vascular causes like arterial hypertension or aortic dissection, or by myocardial damage due to inflammation, fibrosis, intoxication, storage, and neuromuscular disorders leading to left ventricular dilatation, wall thickening, and impaired diastolic and systolic function. The role of microorganisms to induce myocardial damage without affecting the coronary arteries is best established for B. burgdorferi (11, 25, 26). Infection with B. burgdorferi may lead to left ventricular dilatation and systolic dysfunction and should be a differential diagnosis in patients presenting with these findings. As listed in Tables 4 and 5, all four patients with antibodies to B. burgdorferi IgG presented with left ventricular dilatation. Since no myocardial biopsy has been performed for these patients, cardiac borreliosis could not be definitively diagnosed in these patients, but it might be an explanation for their exertional dyspnea and anginal chest pain, especially in the three cases in which no other cause could be assessed (Table 5). Myocardial damage due to myocarditis, leading to heart failure and sudden cardiac death, has been described for infections with C. pneumoniae (28), Ehrlichia (8), R. conorii (16), and Coxiella (14). Cardiac involvement in leptospirosis has been described as ECG abnormalities and pericarditis (27). Myocardial damage by Bartonella and Treponema organisms has been shown in animals (1, 4, 13). Whether these microorganisms cause myocardial damage also in humans is at present unknown.

A limitation of our study is the small number of patients. Therefore, statistical valid tests about associations could not be calculated. Further limitations are the lack of a serologic follow-up and of myocardial biopsies in patients with normal coronary angiograms and serologic findings suggestive of an infection. Additionally, testing for other antibodies to microorganisms known to induce arterial damage, like cytomegalovirus, or myocardial damage, like viruses, other bacteria, fungi, and protozoa, has not been performed.

It is concluded that assessment of the seroprevalence of antibodies to microorganisms known to induce arterial and myocardial damage is not useful to clarify the etiology of exertional dyspnea or anginal chest pain in patients with normal coronary angiograms, since they are similarly distributed as in the general population and do not differ between patients with coronary artery stenosis and with normal coronary angiograms. These findings from a relatively small number of patients have to be confirmed in a larger study.