Potential association between bacterial infections and ischemic stroke based on fifty case-control studies: A systematic review and meta-analysis

Background Stroke is considered as one of the most important concerns in health care centers around the world. By definition there are two types of stroke including ischemic stroke and hemorrhagic stroke. Approximately three-quarters of stroke cases are ischemic strokes, which occur due to several risk factors such as hypertension, obesity, atherosclerosis, diabetes mellitus, osteoarthritis, and inflammatory responses. In recent years, infectious diseases have noticed as a new risk factor for ischemic stroke. Given the importance of the issue, some bacteria that cause chronic infections, especially Chlamydia pneumonia, Helicobacter pylori, Mycoplasma pneumonia, Mycobacterium tuberculosis, and Coxiella burnetii have been considered. Methods In the present meta-analysis, we reviewed 50 case-control studies and assessed the possible association of bacterial infections with the occurrence of ischemic stroke. Results We analyzed the information of 33,978 participants in several nested case-control studies, and ultimately showed that bacterial infections could increase the risk of ischemic stroke. Our results suggest that bacterial infections significantly increase in the risk of ischemic stroke (OR: 1.704; 1.57-1.84 with 95% CIs; p value = 0.01). Conclusions In this meta-analysis, a significant relationship was observed between infection by three bacteria such as C. pneumoniae, H. pylori, and M. tuberculosis with the occurrence of ischemic stroke. Furthermore, due to the similarity between TLRVYK domain in β2-glycoprotein-I and TLRVYK peptide in various of microorganisms, produced antibodies against pathogens interact with β2-glycoprotein-I, hence the cross-reaction phenomenon increases the positive relationship between infectious diseases and ischemic stroke.

The present meta-analysis was performed for the purpose of plausible relationship between ischemic stroke and infection with H. pylori, C. pneumoniae, M. pneumoniae, and Mtb. As well as, the potential role for stroke induction was estimated for each group of infectious agents.

Methods
Literature search strategy Comprehensive systematic search was done through databases such as Scopus, PubMed, Cochrane Library, Embase, and Google scholar up to May 2020. For searching, we used keyword phrases such as "Helicobacter pylori" and "ischemic stroke," "Chlamydia pneumonia" and "ischemic stroke," "Mycoplasma pneumonia" and "ischemic stroke," "Mycobacterium tuberculosis" AND "ischemic stroke," as well as "bacterial infection" and "Ischemic stroke." All published English articles were retrieved without limitation on the date of publication. The search strategy was performed by two authors separately; in case of disagreement, the third author judged and decided.

Study selection criteria
In the present meta-analysis inclusion criteria included: 1) casecontrol studies on the role of bacterial infections in ischemic stroke; 2) studies on the role of infection by H. pylori, C. pneumoniae, M. pneumoniae, and Mtb in ischemic stroke; 3) studies with standard diagnostic methods including ELISA and other immunoassays, conventional microbiology methods, PCR, blotting assay, and urease breath test (UBT); 4) clarity in  the results of included studies. In addition, our exclusion criteria included case-report, letter to editor, review, congress abstract, non-English texts, prospective or cohort, post-stroke infections studies, repetitive results, and unclear studies. The flowchart of included studies is presented in Fig. 1.
Quality assessment and data collection The quality assessment of included studies was evaluated using the Newcastle-Ottawa scale (NOS). Required information included first author, publication year, location of each studies, type of infection agents, diagnostic methods of infection, age, gender, case group, control group, and number of infected cases in both case and control groups (Table 1) .
Quantitative synthesis Data analysis was performed using Comprehensive Meta-Analysis (CMA) software version 2.2 (Biostat, Englewood, NJ). For this purpose, first the frequency of each bacterial infection including H. pylori, C. pneumoniae, M. pneumoniae, and Mtb was measured and according to the event rate (%) was reported for both case and control groups. Next, using the Odds ratio (OR) with 95% Confidence intervals (CIs), potential role of bacterial infection in the occurrence of ischemic stroke was analyzed. Moreover, by Cochran's Q and I 2 statistic parameters, we analyzed heterogeneity of included studies. By our default, the cases with Cochrane Q statistic p < 0.1 and I 2 > 25% were described as cases with high heterogeneity. According to the Dersimonian and Laird method, the random effect model and the fixed effect model were applied in high and low heterogeneity cases, respectively. Finally, Egger's regression was used for estimating asymmetry of funnel plot and also publication bias.

Results
Characterization of included studies After the initial search, 238 documents were identified from 1996-2017, and finally, 50 studies were selected based on inclusion criteria. In a large number of eligible studies, the presence of bacterial infection at the time of acute ischemic stroke was investigated; however, there were also cohort studies that  (Fig. 2).

The potential association between H. pylori infection and ischemic stroke
Of the 50 case-control articles included in this meta-analysis, 18 were conducted on the association between H. pylori infection and the incidence of ischemic stroke. The infection rate in both patient and healthy groups was 63% (60-65 with 95% CIs) and 55% (52-57 with 95% CIs), respectively. In accordance with statistical results, it seems that there is a meaningful relationship between infection by H. pylori and ischemic stroke (OR: 1.64; 1.44-1.87 with 95% CIs; pvalue = 0.001; I 2 = 72.88; Q-value = 59; df = 16; Egger's intercept = 1.87) (Fig. 4).
The potential association between M. pneumonia infection and ischemic stroke Regarding the plausible role of infection by M. pneumonia and occurrence of ischemic stroke, we retrieved only two eligible studies. The incidence rate of infection in both case and control groups was 55% (39-70 with 95% CIs) and 47% (11-86 with 95% CIs), respectively. We did not find any significant relationship between M. pneumonia infection with ischemic stroke (OR: 0.97; 0.12-7.69 with 95% CIs; p-value = 0.98; I2 = 77.94; Q-value = 4.53; df = 1) (Fig. 5). The potential association between M. tuberculosis infection and ischemic stroke Finally, to evaluate the association between infection by Mtb and stroke, we analyzed two studies. Based on statistical analysis, the rate of infection by Mtb in both ischemic and healthy groups was 4% (3.6-4.5 with 95% CIs) and 3% (3.3-4 with 95% CIs), respectively. However, we observed a significant relationship between infection by Mtb and the incidence of ischemic stroke (OR: 1.15; 0.99-1.34 with 95% CIs; p-value = 0.05; I2 = 94.73; Q-value = 18.98; df = 1) (Fig. 6).
In general, in the present study we appraised the potential role of infections by four bacteria including C. pneumoniae, H. pylori, M. pneumoniae, Mtb, and progression to ischemic stroke. In this meta-analysis we demonstrated a meaningful relationship between infection by three bacteria C. pneumoniae, H. pylori, and Mtb with occurrence of ischemic stroke. However, significant heterogeneity was observed between the studies, which in turn led to the unreliability of the current results; moreover, there were differences in items such as bacterial identification methods (i.e. instrument or materials), study design, as well as difference in time-point contributed between included studies. Unfortunately, we did not access to raw data to provide subgroup analysis to reduce heterogeneity. Therefore, more extensive research is needed to validate the current analysis.

Discussion
Stroke is one of the most common cardiovascular disorders, and its occurrence depends on underlying risk factors including hypertension, diabetes mellitus, smoking, hyperlipidemia, atrial fibrillation, atherosclerosis, as well as characteristics such as age, gender, and family history [67]. Depending on the circumstances, the risk factors for the onset of stroke are different, so the mechanism of the increase in the incidence of stroke in young people and its trend towards autumn and winter is not yet fully understood [68]. In recent decades, understanding the role of acute and chronic infections in stroke has received more attention; obviously, infection can lead to inflammation, which in turn causes complications such as the formation of fatty plaques in blood vessels, atherogenic reactions, and changes in host metabolism (Fig. 7) [69].

Keikha and Karbalaei Potential association between bacterial infections and ischemic stroke
According to the literature, the infections caused by pathogens such as C. pneumonia, H. pylori, M. pneumonia, Coxiella burnetii, HIV, HSV 1-2, and CMV are more significant, these infectious microorganisms are often isolated from atherosclerotic plaques [12,70]. On the other hand, induction of CIMT and a high seropositive status in the population with CVD confirm this phenomenon [71]. Today, there is ample evidence of the role of bacterial infections in increasing stroke; for example, infectious endocarditis can lead to embolism and arthritis. Moreover, bacterial meningitis and chronic brucellosis cause vasculitis and thrombosis in cerebral arteries; likewise, rickettsial infections lead to damage of small vascular endothelial cells and eventually ischemic stroke [68].
Induced autoantibodies during infectious diseases are considered pathogenic autoantibodies and potentially interact with the phospholipid binding protein β2-glycoprotein-I (β2GPI), the major autoantigen in APS [72]. Due to the phenomenon of "molecular mimicry," microbes mimic the natural proteins of the host with the help of their chemical structures [73]. In their study, Blank et al. showed that there is a high homology between the TLRVYK hexapeptide in β2GPI and various bacteria (Pseudomonas aeruginosa, Yersinia pseudotuberculosis, and Streptomyces lividans), viruses (CMV, polyoma virus, and adeno virus-40), yeasts (Saccharomyces cerevisiae), and parasites (Schistosoma mansoni), which in turn induces pathogenic H-3 anti-β2GPI mAb against β2GPI [74,75]. In the present study using statistical analysis of fifty case-control studies, we found that there is a significant relationship between bacterial infection and ischemic stroke cases (OR: 1.7; CI: 1.5-1.8).
C. pneumonia is a Gram-negative intracellular bacterium that was first introduced in 1980s. More than half of the world's population is infected with this bacterium; this fact has been confirmed by serological evidence [76,77]. In many studies, researchers have isolated C. pneumonia from carotid plaques, atherosclerotic plaques, and circulating leukocytes [77]. Sander et al. showed that eliminating C. pneumoniae infection could prevent the progression of CIMT [78]. Clinical trial studies have also suggested that treatment of this infection can potentially reduce vascular lesions in patients [79]. In our study the rate of infection by C. pneumonia in both case and control groups was measured 57% and 36% respectively. Also, we showed that there is a meaningful relationship between infection by this bacterium and occurrence of ischemic stroke (OR: 2.14; CI: 1.9-2.3).
H. pylori is a Gram-negative, helical, and microaerophilic bacterium that colonizes almost half of the world's population in the gastric mucosa [80]. The rate of infection by this bacterium is higher in developing countries than in developed countries, accordingly, in some parts of Africa the infection with this pathogen reaches 100% [81,82]. This bacterium is considered as a causative agent in disorders such as chronic gastritis, gastric ulcer, as well as gastric cancer; extragastrointestinal diseases related to this pathogen have attracted much attention, in which the association between H. pylori infection and CVD is well known [83,84]. In addition to isolating these bacteria from atherosclerotic plaques, their infection causes disorders such as insulin resistance, dyslipidemia, hypertension, and alteration in metabolism, all of which describe the potential role of H. pylori infection in increasing ischemic stroke, particularly nonembolic ischemic stroke [85,86]. Statistically, infection with this pathogen was measured at 63% and 55% in ischemic stroke patients and healthy individuals, respectively (OR: 1.6; CI: 1.4-1.8).
M. pneumonia is a respiratory pathogen that despite a poor understanding of its pathogenicity, many people have anti-M.pneumonia antibodies (IgG and IgM). Recent studies have shown its role in extra-pulmonary manifestations such as musculoskeletal, gastrointestinal, neurological, dermatologic, hematologic, and cardiovascular complications [87,88]. According to the literature, about 0.1% of patients infected with M. pneumonia develop neurological disability during their lifetime [89]. Vasculopathic lesions following this bacterial infection indicate the role of M. pneumonia in increasing the risk of ischemic stroke [68,90]. Two studies in this meta-analysis were related to the role of M. pneumonia in ischemic stroke susceptibility; infection by this bacterium was 55% and 47% in both case and control groups, respectively. Also, based on statistical analysis, no significant association was found between infection by M. pneumonia and ischemic stroke (OR: 0.97; CI: 0.12-7.6). However, the small number of studies may affect the present results, as only two studies had been performed on this bacterium. In addition, high heterogeneity causes instability of the results and we need more studies to confirm these findings.
Mtb is a life-threatening pathogen, and although this bacterium is commonly identified as a major cause of pulmonary tuberculosis, it also causes extra-pulmonary manifestations [91,92]. In recent years the role of bacterium in promoting neurological manifestation has been demonstrated, so that following arterial invasion, malignant vasculitis can be occurred during the tubercular meningitis [68]. Salindri et al. conducted a cohort study to show the effects of Mtb infection on chronic non-communicable disease; they found that previous tuberculosis (TB) infection could significantly increase susceptibility to ischemic stroke [93]. Our results also confirmed the possible association between TB infection and an increased risk of ischemic stroke.
Coxiella burnetii is an intracellular and Gram-negative coccobacillus bacterium which causes Q-fever, a zoonosis disease; Q-fever is endemic worldwide, especially in European countries e.g. Spain, France, and Germany [94]. Based on the findings of Vinacci et al., Coxiella burnetii is one of the main bacterial isolates in patients with infective endocarditis (IE) and acute ischemic stroke (AIS) [95]. During primary infection, Coxiella burnetii induces high levels of antiphospholipid antibodies, especially IgG anticardiolipin antibodies (IgG aCL), which in turn lead to antiphospholipid syndrome (APS); infectious IgG aCL are associated with several complications such as fever, thrombocytopenia, valvular heart disease, as well as chronic endocarditis [96][97][98]. In addition, serological markers indicate that there is a significant relationship between Coxiella burnetii infection and CVD in the elderly people [99,100].
Nevertheless, our study had several limitations which we must mention: 1) the small number of included studies; 2) lack of access to raw data to perform modulatory analysis to describe significant heterogeneity; 3) the effects of mixed infections are likely to be underestimated in qualified studies; 4) asymmetry of funnel plot also suggests the presence of significant publication bias within the included studies; 5) there was also difference in diagnostic method, study design, studies timepoint, population ethnicity and location of included studies that actively contributed in heterogeneity of the included studies. In our study, a large number of eligible studies were crosssectional that assessed the bacterial infections at the time of ischemic stroke, while, cohort studies could better deliberate the clear association between bacterial infection and susceptibility to ischemic stroke. Therefore, to confirm the results of the present study, we need more comprehensive studies.

Conclusion
In the present study, the information of two nested case-control studies about the role of Mtb in ischemic stroke was evaluated. The rate of infection by this pathogen in both cases and control groups was 4% and 3%, respectively, and we found a significant association between mycobacterial infection and ischemic stroke (OR: 1.1; CI: 0.99-1.34). We assessed the relationship between bacterial infections and the development of ischemic stroke. Nevertheless, due to limitation in results, we could not evaluate the role of pre-existing risk factors in our research. Overall, for understanding the role of bacterial infections in ischemic stroke, it is better to perform a comprehensive study about the association between bacterial infections and traditional ischemic stroke risk factors, CIMT, atherosclerosis, and cardiovascular risk factors such as LDL and HDL. Our results indicate the need for additional longitudinal investigations to determine the impact of infectious disease on the risk of ischemic stroke; such studies will require substantial follow-up time and control subjects. We recommend further larger cohort studies to determine bacterial infections and their determining role in ischemic stroke. As we noted above, TLRVYK domain in β2GPI is the main peptide in cardiolipins structures and is homologous with TLRVYK peptide in various bacteria, viruses, yeasts, and parasites. Pathogenic autoantibodies (H-3 anti-β2GPI mAb) produced against infectious microorganisms cross-react with this peptide in β2GPI, thus may lead to ischemic stroke.

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