The Relationship Between Seropositivity Against Chlamydia pneumoniae and Stroke and its Subtypes in a Latvian Population

Background and Objective. Serological evidence of infection with Chlamydia pneumoniae has been associated with cardiovascular diseases, but the relationship with stroke and its risk factors remains not completely understood. The aim of this study was to determine whether serological evidence of infection with Chlamydia pneumoniae was associated with the risk of ischemic stroke and any of investigated stroke subtypes. Material and Methods. Confirmed stroke cases (n=102) were compared with genderand agematched control patients (n=48). The patients with stroke were divided into 3 groups according to the TOAST criteria: atherothrombotic (n=36), cardioembolic (n=47), and of undetermined etiology (n=19). Plasma levels of IgG antibodies to Chlamydia pneumoniae were measured by enzymelinked immunosorbent assay. Results. There was a significant association between seropositivity to Chlamydia pneumoniae and stroke. Anti-Chlamydia pneumoniae IgG antibodies were detected in 64 case patients (62.7%) and 17 control patients (35.4%) (χ2=9.8; df=1; P=0.002). IgG seropositivity to Chlamydia pneumoniae was linked to all the analyzed etiological subtypes of stroke. Conclusion. This study showed that IgG seropositivity to Chlamydia pneumoniae was associated with stroke and all the analyzed etiological subtypes of stroke.


Introduction
Vascular diseases, undoubtedly, are one of the major causes of mortality worldwide, whereas stroke remains a leading cause of long-term disability.Mortality from stroke in Latvia is rather high; moreover, it is one of the highest in the region comparing with the neighboring countries (1).
Atherosclerosis is one of the main risk factors for cerebrovascular diseases, not only directly affecting intra-and extracranial blood vessels, but also affecting other organs and systems thus increasing disease burden and costs and lowering quality of life.
Chronic inflammatory reaction is hypothesized to play a role in the development of atherosclerotic process; furthermore, it might be considered as an independent risk factor for stroke (2).
C. pneumoniae has been linked to atherosclerosis (3) and vascular accidents.The relationship between C. pneumoniae and stroke has not been confirmed by all trials; therefore, the data are controversial.Several serologic studies have established a link between seropositivity and acute cerebrovascular accident (4-8), while others have not demonstrated any association (9)(10)(11).
The aim of this study was to determine whether serologic evidence of infection with C. pneumoniae was associated with the risk of ischemic stroke and any of investigated stroke subtypes.

Material and Methods
A retrospective study was carried out.It was approved by the hospital's Ethics Commission.All the case patients (n=102) were patients with acute ischemic stroke admitted to the Paula Stradins Clinical University Hospital from October 2007 to March 2009.Stroke was defined as a focal (or at the time global) neurological impairment of sudden onset, and lasting more than 24 hours (or leading to death), and of presumed vascular origin according to the WHO definition.
Eligible cases were enrolled if they had a firstever stroke or recurrent cerebral infarction.Patients with cerebral infarction secondary to other diseases, lacunar stroke, past medical history of oncologic or chronic inflammatory diseases, and severe impairment of renal function were excluded from the study.All the patients were categorized into 3 groups using the Trial of ORG 10172 in Acute Stroke Treatment (TOAST) criteria (12): atherothrombotic, cardioembolic, and of undetermined etiology.In addition, the study questionnaire included the evaluation of neurological status on admission, modified Rankin scale, stroke localization (middle cerebral artery [MCA]) anterior cerebral artery [ACA] and vertebrobasilar [VB] territories), and stroke risk factors.Computed tomography was used as a routine neuroimaging method (100% of study subjects), while magnetic resonance imaging was performed if required.Control patients were matched to case patients by age and gender (n=48; 26 men and 22 women; mean age, 64.3 years [SD, 11.8]; age range, 42 to 82 years) and were patients with nonvascular diseases, predominantly noninflammatory lower back pain, admitted to the P. Stradins Clinical University Hospital.The ratio of control patients to case patients was about 1:2.Stenosis of brachiocephalic vessels exceeding 60% detected by Doppler ultrasound using flow velocity analysis was considered significant.Overweight was defined as body mass index greater than 25 kg/m 2 according to the WHO criteria.
To detect IgG to C. pneumoniae, Novagnost ™ (Germany) ELISA system was used.A value of 8 IU/ mL was considered as indicative of positive result.Both semiquantitative and qualitative tests were employed for the present study.
Statistical Analysis.SPSS 16.0 for Windows was used for data processing and analysis.To compare the mean values, both the analysis of variance (ANOVA) and the t test were used.Frequency expressed in % was obtained by applying multifactorial (r×c) frequency tables.Frequency distribution was detected by the Pearson χ 2 and Fisher tests using "Statcalc," but covariance was calculated by applying Pearson correlation coefficient.Differences were considered significant at P≤0.05. 1.

Demographic characteristics of the recruited subjects are shown in Table
There were no significant age (t=0.806;P=0.422) and gender (χ 2 =0.426; df=1; P=0.514) differences comparing the case and control groups (Table 1).The mean age of patients with atherothrombotic stroke was 63.19 years (SD, 11.3); with cardioembolic stroke, 69.9 years (SD, 8.8); and with stroke of undetermined etiology, 60.7 years (SD, 11.9).The difference in the mean age of stroke patients according to the subtypes was significant (F=4.631;P=0.004) (Table 2).
Table 3 illustrates data on the main risk factors for stroke.Arterial hypertension was found to be the most common risk factor detected in 83.4% of case patients (n=86).
Seropositivity against C. pneumoniae was documented in 64 (62.7%) of the 102 patients and only in 17 (35.4%) of the 48 controls (χ 2 =9.8; df=1; P=0.002).The mean serum antibody level in the patient group was slightly higher compared with the control group, but the difference was not significant (9.89±4U vs. 8.8±4.4U; t=1.465,P=0.15).Analysis of each group with different stroke subtype revealed IgG seropositivity against C. pneumoniae in 58.33% of atherothrombotic patients, and this percentage was significantly greater than that of seropositive patients in the control group (35.42%) (χ 2 =4.36;P=0.037).In the groups of cardioembolic stroke and stroke of undefined etiology, 65.96% and 63.16% of patients, respectively, were found to be seropositive, and these percentages were significantly greater as compared to the control group as well (χ 2 =8.86,P=0.003; and χ 2 =4.27,P=0.039; respectively).
Lipid profile and its association with C. pneumoniae IgG status were retrospectively analyzed.For this purpose, patients with stroke were divided into two

Discussion
The link between C. pneumoniae and an acute stroke has not been established in all studies, which have looked at this issue.Furthermore, various studies employed different methods.Some trials that employed serologic methods have demonstrated a link between seropositivity to C. pneumoniae and an acute cerebrovascular accident (4-8), whereas others have not (9)(10)(11).Moreover, some studies have confi rmed the presence of C. pneumoniae in the atherosclerotic plaque by molecular genetic methods (13)(14)(15)(16)(17), but a considerable number of studies have not reported any association (18)(19)(20)(21).Likewise, the relationship between bacteria and atherosclerotic changes in intracerebral blood vessels is not straightforward.A study from Netherlands using polymerase chain reaction (PCR) was unable to detect C. pneumonia in large intracerebral vessels (22); whereas the study that examined the atherosclerotic middle cerebral artery was able to detect it by nested PCR in 5 of the 15 atherosclerotic arterial samples and none of the control tissues (23).Despite a plausible link between bacteria and atherosclerosis described in a review by Watson and Alp (24), the situation with stroke might be more complicated due to a possibly weaker link between an acute ischemic cerebrovascular event and atherosclerosis if compared to coronary artery disease and myocardial infarction.Consequently, it may be that no certain bacteria are associated with stroke, but perhaps infectious burden of common pathogens actually might be linked to stroke (25) or carotid atherosclerosis (26).
Stroke is a heterogenous disease that is classifi ed into several subtypes of different etiology, and associations between subtypes and C. pneumoniae has not been thoroughly investigated yet.Moreover, results of the studies appear to be controversial, not only due to the diffi culties in the detection of C. pneumoniae, but also due to differences in the defi nition for stroke subtypes among researchers.
In the present study, an association between seropositivity to C. pneumoniae and a stroke was observed.Unfortunately, the present study has some limitations: certain stroke subtypes, such as lacunar and a stroke associated with other pathologies, were excluded, and the sample size was small.However, our study showed evidence for an association between C. pneumoniae and investigated subtypes of stroke.
Arterial hypertension, coronary artery disease, peripheral artery disease, etc. are well-known and wellstudied classic risk factors for stroke (27).Dyslipidemia is one of the classic risk factors, which have been demonstrated to have some link with C. pneumoniae seropositivity in some studies (28)(29).Currently, there is room for more studies to clarify an interaction between infectious agents and dyslipidemia, whether C. pneumoniae is just an innocent bystander or an active player.This study showed a signifi cant link between C. pneumoniae seropositivity and elevated serum LDL level.It might be worth to investigate further a role of microorganisms in lipid profi le of atherogenesis.
Population health, effi cient use of state budget, and quality of life are relevant questions requiring a continuing search for effective solutions of the stroke problem.However, there are many problems to be solved to confi rm the infection theory and its role in stroke etiology.Every new research is a next step in assembling this multi-layer jigsaw puzzle and fi nding a solution to the complicated problem.

Conclusions
Our serological study showed an association between the presence of C. pneumoniae antibodies and stroke in the Latvian population.In addition, it demonstrated the signifi cance of C. pneumoniae seropositivity for several major stroke subtypes and correlation with elevated serum low-density lipoproteins level.To support atherogenic characteristics of Chlamydia pneumoniae, we need large prospective cohort studies and randomized controlled interventional trials, which would combine serologic and nonserologic methods for confi rmation of chronic infection in selected population.

Statement of Conflict of Interest
The authors state no confl ict of interest.

Table 1 .
Demographic Characteristics of the Study Population

Table 2 .
Demographic Characteristics of Case Patients by Stroke Subtype

Table 3 .
Risk Factors by Study Groups

Table 4 .
C. Pneumoniae Serum Antibody IgG Level by Different Groups Seropositivity Against Chlamydia pneumoniae and Stroke in a Latvian Population