Association of Chlamydial Infection With Cerebrovascular Disease
Background and Purpose Recent studies suggest an association of coronary heart disease and carotid atherosclerosis with Chlamydia pneumoniae infection. We investigated the frequency of chlamydial seropositivity and specific circulating immune complexes in patients with recent cerebrovascular disease.
Methods Specific antibodies to C pneumoniae in serum were measured by the microimmunofluorescence test in 58 consecutive patients (aged 18 to 50 years) with ischemic infarction (n=39) or transient ischemic attacks (n=19) and in 52 hospital control subjects without vascular disease, matched for sex, age, time, and locality.
Results Twenty-seven patients (46.6%) and 12 control subjects (23.1%) had raised IgA titers ≥1:16 (P=.018). IgG titers ≥1:32 were measured in 74.1% of the patients and 77% of control subjects (P=.623). Specific IgG antibodies in circulating immune complexes, which were isolated by polyethylene glycol precipitation, were elevated ≥1:8 in 24.1% of the patients and 7.7% of control subjects (P=.047). With the use of a conditional logistic regression model, the odds ratios were 1.70 (95% confidence interval [CI], 1.13 to 2.58) for elevated IgA titers, 1.91 (95% CI, 1.06 to 3.47) for the presence of immune complexes, and 1.96 (95% CI, 1.00 to 3.82) for the presence of both factors. After adjustment for the vascular risk factors hypertension, age, sex, and migraine, the odds ratios were 1.71 (95% CI, 1.08 to 2.70), 2.00 (95% CI, 1.07 to 3.76), and 2.20 (95% CI, 1.09 to 4.41), respectively.
Conclusions We conclude that chronic infection with C pneumoniae is associated with an increased risk of stroke and transient ischemic events.
Chlamydiae are obligatory intracellular, gram-negative bacteria that are known for their ability to cause chronically progressive infection with severe sequelae such as blindness and infertility (Chlamydia trachomatis). Ten years ago a new human pathogen of the Chlamydia species was detected: Chlamydia pneumoniae.1 C pneumoniae frequently causes community-acquired respiratory infections such as sinusitis, pharyngitis, and pneumonia. There are epidemics due to C pneumoniae every 5 to 7 years in northwestern countries, with a 50% to 70% prevalence of seropositivity in middle-aged adults and most of the adults being infected 2 to 3 times during their lifetime.2 Recently, several laboratories pursued the hypothesis that C pneumoniae may cause chronically progressive infection similar to other Chlamydia species and that chronic C pneumoniae infection may be associated with atherosclerosis.3 4 The association was supported by raised IgG and/or IgA titers against C pneumoniae in patients with myocardial infarction, coronary heart disease,3 5 6 and asymptomatic carotid atherosclerosis7 and the finding of C pneumoniae by immunocytochemistry and polymerase chain reaction in coronary and aortic atheroma but not in normal arteries.8 9 10 As additional evidence for chronic C pneumoniae infection, C pneumoniae–specific circulating immune complexes were found more frequently in patients with coronary heart disease than in control subjects.11 The aim of the present study was to determine whether chlamydial infection is associated with CVD. The study was restricted to patients younger than 50 years, in whom other risk factors were missing or were less prominent.
Subjects and Methods
We investigated 58 patients, 39 with cerebral infarction and 19 with TIA, who were admitted consecutively to the Department of Neurology, Klinikum Grosshadern, University of Munich, from September 1993 to November 1994. Fifty-two patients of the department hospitalized during the same time period were matched control subjects without vascular disease with the diagnoses epilepsy (n=25), disc herniation, (n=7), benign paroxysmal positional vertigo (n=4), head trauma (n=4), polyneuropathy (n=4), multiple sclerosis (n=2), dystonia (n=1), myasthenia (n=1), aqueductal syndrome (n=1), schizophrenia (n=1), and depression (n=1). Both patients and control subjects were from Munich or surrounding areas. The study was limited to subjects and control subjects aged 50 years or younger. With the use of duplex sonography, transesophageal echocardiography, CT imaging, and angiography, the likely cause of stroke was classified as cardioembolic in 20% of our patients, thrombotic in 30%, and microangiopathic in 5%. In 45% of the patients the probable cause of ischemia was not identified. Blood samples for chlamydial serology were usually taken within 1 week after admission to the hospital (mean, 4.4±3.97 days; range, 1 to 15 days) and stored at −80°C until analysis. All other laboratory tests were done in the clinical routine, including ESR and CRP as markers of acute infection. A standardized interview was performed with patients and control subjects to evaluate risk factors for vascular disease (history of high blood pressure, diabetes, migraine, oral contraception, and smoking habits) and symptoms of infection before admission (fever, headache, cough, hoarseness, sore throat, chills, myalgia, sweating, bronchitis, pharyngitis, arthritis, and gynecological or urological disturbances).
Chlamydial studies were performed in a blinded fashion; sera from both patients and control subjects were included in all test series. Sera were tested by MIF for the presence of specific IgG, IgA, and IgM antibodies to C pneumoniae and C trachomatis with a modification of the method of Wang and Grayston.12 Three C pneumoniae strains were used as antigens: IOL 207, Parola, and Kajaani 6. Starting at 1:16, twofold dilutions of sera were incubated at 36°C with preparations of elementary bodies of the different Chlamydia strains fixed to microscopy slides previously. After the incubated slides were washed with phosphate-buffered saline (pH 7.4) to remove antibodies not bound to the antigen, fluorescein isothiocyanate–labeled conjugates against human immunoglobulins were used with Amido Schwarz as counterstain. The highest dilution of sera leading to a homogeneous sharp demarcation of elementary bodies in fluorescence microscopy was considered positive. Since MIF results between the different strains yielded a very high correlation (0.86 to 0.99) and protein profiles of various C pneumoniae preparations have been shown to be identical,13 Kajaani 6 was selected as a representative C pneumoniae antigen. To avoid the interference of IgG and rheumatoid factor, which results in false-positive IgM levels14 and competition between IgG and IgA,15 sera for both IgM and IgA determinations were treated with GullSORB reagent (Gull Laboratories) to remove interfering IgG antibodies by immunoprecipitation with anti-human IgG.
Circulating immune complexes were isolated by polyethylene glycol precipitation according to the modified method of Schutzer et al,16 as previously described in detail. The dissociated immune complexes were then analyzed in MIF for the presence of IgG antibodies against Chlamydia at twofold dilutions starting at 1:2.
Data were computed with SPSS (Statistical Package for the Social Sciences, SPSS Inc). Rates and proportions were compared with the χ2 test with Yates' correction. When the minimum estimated expected value was less than 5, Fisher's exact test was used. Quantitative variables were analyzed with the Mann-Whitney test. To analyze the association of chlamydial antibody prevalence and CVD, unadjusted and adjusted odds ratios and the corresponding 95% CIs were calculated with a logistic conditional regression model, controlling for age, sex, hypertension, and migraine in the patient's history. P<.05 was considered statistically significant.
The demographic characteristics and distribution of risk factors for CVD in patients and control subjects are presented in Table 1⇓. Hypertension was reported by 31% (n=18) of CVD patients and 3.8% (n=2) of the control subjects (P=.0002), and the frequency of migraine was 15.5% (n=9) versus 1.9% (n=1), respectively (P=.032). To evaluate potential confounding, both factors were used as covariates together with age and sex in the logistic regression analysis. In regard to all other risk factors evaluated as well as symptoms of infection before the event, there was a distinct tendency in favor of the CVD group, although the level of significance was not reached. More patients than control subjects had slightly elevated leukocytes (10 g/L) and subfebrile temperatures (37°C to 38°C), but patients and control subjects did not differ in ESR or CRP.
The geometric means of IgA antibody titers to C pneumoniae and specific IgG in circulating immune complexes were significantly higher in patients than in control subjects. Analyses performed separately for the two groups (cerebral infarction and TIA) showed a similar distribution of antibody titers in both subgroups, with only discretely higher values in the cerebral infarction group. The number of cases did not permit further statistical evaluation of subgroups (Table 2⇓). IgA antibody titers ≥1:16 against C pneumoniae were found in 46.6% (n=27) of patients and 23.1% (n=12) of control subjects (P=.018). Immune complexes containing C pneumoniae–specific IgG at titers ≥1:8 were measured in 24.1% (n=14) of the patients and in 7.7% (n=4) of the control subjects (P=.047). The seroprevalence of elevated IgG titers (≥1:32) against C pneumoniae was high in patients and control subjects, at 74.1% (n=43) and 76.9% (n=40), respectively (P=.623). IgM titers ≥1:40 against C pneumoniae occurred in 5.2% (n=3) of the patients and in none of the control subjects (P=.297). Antibodies against C trachomatis were found in 6.9% (n=4) of patients and none of the control subjects (P=.122).
Odds ratios indicating an association with an increase in the risk of CVD were 1.70 for elevated IgA antibodies (95% CI, 1.13 to 2.58), 1.91 for immune complexes containing specific IgG at titers ≥1:8 (95% CI, 1.06 to 3.47), and 1.96 (95% CI, 1.00 to 3.82) for the presence of both. After adjustment for age, sex, hypertension, and migraine, the corresponding values were 1.71 (95% CI, 1.08 to 2.70), 2.00 (95% CI, 1.07 to 3.76), and 2.20 (95% CI, 1.09 to 4.41), respectively (Table 3⇓). The factors elevated cholesterol or triglycerides in serum, smoking habits, and body mass index did not change these findings when added into the regression model.
In this study, IgA antibodies against C pneumoniae in serum and circulating immune complexes containing C pneumoniae–specific IgG antibodies were more frequent in patients with recent cerebral ischemia than in control subjects. The presence of IgA antibodies and specific immune complexes were independently associated with the risk factors of CVD in the logistic regression analysis. There was no confounding by commonly recognized vascular risk factors, including hypertension, migraine, serum cholesterol and triglycerides, smoking, and overweight. Therefore, there may be a risk enhancement for stroke by C pneumoniae infection, as has already been suggested for coronary artery disease and carotid atherosclerosis.
It is known from previously published populations in northwestern countries that the seroprevalence for positive IgG titers against C pneumoniae is high among adults, suggesting that most adults are infected one to several times during their life.2 17 In accordance, the present study shows positive IgG titers in more than two thirds of patients as well as control subjects. The high seroprevalence in both groups argues against the possibility of a selection bias in one of the groups, which may occur in a small case-control study such as the present one, although patients and control subjects were recruited from the same geographic area and the same time period. In neither of the two groups were symptoms of acute C pneumoniae infection overt, such as respiratory disease, fever beyond 38°C, or inflammatory signs in the blood. Moderate increases in the leukocyte count and subfebrile temperatures in the CVD group, in the absence of increased ESR or CRP levels, may be a response to ischemia rather than evidence of infection. The infrequency of acute C pneumoniae infection is supported by the few patients in whom C pneumoniae IgM titers were found. The serological pattern of increased IgA titers and specific IgG-containing immune complexes has been suggested to indicate chronic persistence of active infection, while IgG titers in the absence of IgA titers or immune complexes may be a serological marker of an older, inactive infection.3 11 If this hypothesis is correct, chronically active rather than acute or chronic but inactive C pneumoniae infection may increase the risk of CVD. Unless there is further clinical evidence for a persistence of C pneumoniae infection, however, the hypothesis remains speculative.
It is possible that the specific immune complexes in circulating blood and raised C pneumoniae titers result from immunologic processes triggered by cerebrovascular damage rather than being associated with the generation of vascular occlusion. To exclude this possibility, prospective cohort studies are needed in which patients with elevated titers are investigated for subsequent vascular occlusive disease. Although these studies do not exist for CVD, raised IgA and the presence of immune complexes were shown to be associated with an increased risk of symptomatic coronary artery disease within the subsequent 6 months in the prospective Helsinki Heart Study.18
The mechanisms underlying vascular occlusion in C pneumoniae infection were not evaluated in this study. C pneumoniae has been shown to multiply in alveolar macrophages and in endothelial cells in culture.19 The presence of circulating immune complexes in this and in previous studies suggests that C pneumoniae actually gains access to the circulation in humans, possibly by invading cells in the vessel wall. C pneumoniae in the wall of atherosclerotic but not normal extracerebral arteries in humans has been demonstrated by immunocytochemical stain and polymerase chain reaction.8 9 10 In addition to invasion and destruction of vessel wall cells, C pneumoniae and its lipopolysaccharide cell wall component are believed to induce tumor necrosis factor, interleukin-2, lipoproteins, and tissue factor, which contribute to a procoagulant state.20 21 22 Direct atherosclerotic occlusion of cerebral vessels, therefore, is only one of several possible mechanisms for stroke. It is speculated that C pneumoniae infection also enhances the risk of embolic stroke (approximately 20% of strokes in this study) or stroke of undetermined etiology (45% in this study).
This study adds another piece to previous evidence that infection is an important risk factor for ischemic brain infarction.23 24 Whereas the previous studies emphasized the significance of acute infections, mostly respiratory and of bacterial origin, this is the first investigation that relates CVD to a chronic infection. The merit of screening patients for elevated C pneumoniae IgA titers and specific immune complexes in the primary and secondary prevention of stroke depends on whether treatment strategies such as antibiotic therapy or platelet inhibitors can reduce the number of subsequent ischemic episodes.
Selected Abbreviations and Acronyms
|ESR||=||erythrocyte sedimentation rate|
|TIA||=||transient ischemic attack|
This study was supported by Sanofi-Winthrop GmbH. The study was performed as part of the dissertation of M.L.J.W. at the Faculty of Medicine, Ludwig-Maximilians University, Munich. We thank J. Benson for assistance in the preparation of the manuscript.
- Received July 8, 1996.
- Revision received August 13, 1996.
- Accepted August 13, 1996.
- Copyright © 1996 by American Heart Association
Leinonen M. Pathogenetic mechanisms and epidemiology of Chlamydia pneumoniae. Eur Heart J. 1993;14(suppl K):57-61.
Grayston JT. Chlamydia in atherosclerosis. Circulation. 1993;87:1408-1409.
Kuo CC, Shor A, Campbell LA, Fukushi H, Patton DL, Grayston JT. Demonstration of Chlamydia pneumoniae in atherosclerotic lesions of coronary arteries. J Infect Dis. 1993;167:841-849.
Campbell LA, O'Brien ER, Cappuccio AL, Kuo CC, Wang SP, Stewart D, Patton DL, Cummings PK, Grayston JT. Detection of Chlamydia pneumoniae TWAR in human coronary atherectomy tissues. J Infect Dis. 1995;172:585-588.
Kuo C-C, Grayston JT, Campbell LA, Goo YA, Wissler RW, Benditt EP. Chlamydia pneumoniae (TWAR) in coronary arteries of young adults (15-34 years old). Proc Natl Acad Sci U S A. 1995;92:6911-6914.
Linnanmäki E, Leinonen M, Mattila K, Nieminen MS, Valtonen V, Saikku P. Chlamydia pneumoniae–specific circulating immune complexes in patients with chronic coronary heart disease. Circulation. 1993;87:1130-1134.
Wang SP, Grayston JT. Microimmunofluorescence serological studies with the TWAR organism. In: Oriel JD, Ridgeway G, Schachter J, Taylor Robinson D, Ward M, eds. Chlamydial Infections. Cambridge, Mass: Cambridge University Press; 1986:329-332.
Iijima Y, Miyashita N, Kishimoto T, Kanamoto Y, Soejima R, Matsumoto A. Characterization of Chlamydia pneumoniae species-specific proteins immunodominant in humans. J Clin Microbiol. 1994;32:583-588.
Verkooyen RP, Hazenberg MA, Van Haaren GH, Van den Bosch JM, Snijder RJ, Van Helden HP, Verbrugh HA. Age-related interference with Chlamydia pneumoniae microimmunofluorescence serology due to circulating rheumatoid factor. J Clin Microbiol. 1992;30:1287-1290.
Jauhiainen T, Tuomi T, Leinonen M, Kark JD, Saikku P. Interference of immunoglobulin G (IgG) antibodies in IgA antibody determinations for Chlamydia pneumoniae by microimmunofluorescence test. J Clin Microbiol. 1994;32:839-840.
Grayston JT, Kuo CC, Campbell LA, Benditt EP. Chlamydia pneumoniae, strain TWAR and atherosclerosis. Eur Heart J. 1993;14(suppl K):66-71.
Saikku P, Leinonen M, Tenkanen L, Linnanmäki E, Ekman MR, Manninen V, Mänttäri M, Frick MH, Huttunen JK. Chronic Chlamydia pneumoniae infection as a risk factor for coronary heart disease in the Helsinki Heart Study. Ann Intern Med. 1992;116:273-278.
Saikku P. Chlamydia pneumoniae infection as a risk factor in acute myocardial infarction. Eur Heart J. 1993;14(suppl K):62-65.
Höiby N, Döring G, Schiötz PO. Antibody and immune complexes induce tissue factor production in human endothelial cells. J Immunol. 1986;40:29-53.
Nurminen M, Leinonen M, Saikku P, Mäkelä PH. The genus specific antigen of Chlamydia: resemblance to the lipopolysaccharide of enteric bacteria. Science. 1983;220:1279-1281.
Syrjänen J, Valtonen VV, Iivanainen M, Kaste M, Huttunen JK. Preceding infection as an important risk factor for ischaemic brain infarction in young and middle aged patients. Br Med J Clin Res Ed. 1988;296:1156-1160.
Grau AJ, Buggle F, Heindl S, Steichen-Wiehn C, Banerjee T, Maiwald M, Rohlfs M, Suhr H, Fiehn W, Becher H, Hacke W. Recent infection as a risk factor for cerebrovascular ischemia. Stroke. 1995;26:373-379.