Infections have long been suspected of playing a role in atherosclerotic diseases. Infection by Helicobacter pylori (H. pylori) causes one of the most common chronic infections, occurring in approximately half of the world's population. H. pylori infection is common in the gastric mucosa, which is approximately classified as three types: type I includes highly virulent strains, which creates cytotoxin-associated gene-A (CagA) toxin and VacA toxin, which is strongly related to CagA, one of the Cag genes.1 Recent studies revealed the presence of H. pylori, especially infective CagA-seropositive strains, in atherosclerotic plaques2,3 and showed H. pylori's association with atherosclerotic diseases. There is now clear evidence that the cytotoxic strains of H. pylori bearing the CagA have a greater potential for eliciting a systemic immune response and are associated with increased inflammation in the development of atherothrombosis.4 Several subsequent case-control and cohort studies assessed the association of CagA status with atherosclerotic diseases. Patients with atherothrombotic disease seemed more likely to be infected by the CagA-seropositive strains of H. pylori than healthy subjects. However, studies that served to evaluate the relation of CagA status and atherosclerotic diseases risk have not produced consistent results, possibly because of the lack of adequate statistical power, selection bias, or population diversity. A meta-analysis may avoid some of these methodological difficulties. In this review, we systematically identified and combined the relevant population-based present studies of the association of CagA-seropositive strains of H. pylori with the risk of ischemic stroke and coronary heart disease to produce a comprehensive measure of the association through a meta-analysis.
The Medline, the Cochrane Collaboration database, EMBASE were used as major databases and the literature search was performed for articles entered from 1990 through October 2006; the databases of CBMdisc and CNKI were also used from 1990 to July 2007. Search key words from MESH index included ‘Helicobacter pylori’ ‘CagA’ and ‘cytotoxin-associatedgene-A’ combined with the words ‘atherosclerosis’, ‘cerebral infarction’, ‘coronary artery atherosclerotic disease’, ‘ischemic stroke’, ‘coronary heart disease’, ‘ischemic cerebrovascular disease’, and ‘atherothrombotic cerebral infarction’. The languages were restricted to Chinese and English. In order to reduce publication bias, we collected data by contacting investigators to request unpublished data. Though it was virtually impossible to obtain all the unpublished data, we still conducted a manual search from references of the papers retrieved, which were important academic conference articles and important Masters’ and Doctorials' dissertations. For studies reiterating the same study, only the most recent study was included. Authors were contacted to ensure that possibly overlapping study populations were included only once. If the authors had not responded, then only the most recent or largest study was included, while duplicate publications were excluded. The abstracts, review articles, editorials, commentaries and book chapters were excluded.
We restricted our review to the case-control studies on infective, chronic CagA-seropositive strains of H. pylori, involving the risk of ischemic stroke and coronary heart disease. Thus, the inclusion criteria for the studies were (1) cases and controls must be similar for all important features except those for the disease in question, or they are matched, or adjustment is performed in the calculation of odds ratios; (2) demographic characteristics of cases and controls are similar; (3) the confirmed diagnosis of the atherosclerotic diseases must be valid; (4) information about the exposure must be gathered in the same way as for cases and controls; (5) validated testing tool(s) for detecting CagA status and H. pylori infection must be used and (6) the detection of CagA status and H. pylori infection must be performed blindly. All identified publications were independently evaluated and selected by two investigators for compliance with these criteria.
A meta-analysis was carried out to assess the relationship between the CagA-seropositive strains of H. pylori and ischemic atherosclerotic disease. Data analysis was performed using the RevMan 4.2. The studies were pooled by averaging the ORs and 95% CIs with 2-tailed P values through a fixed-effects model, using the Mantel-Haenszel approach when no heterogeneity was observed among the studies. When any evidence of heterogeneity (P <0.1 was considered evidence of heterogeneity) among studies was detected, a random-effects model was adopted for a combination of all studies. Heterogeneity among studies was tested by the χ2 statistic obtained by summing up the weighted squares of the deviations of each estimate from the pooled estimate.
To check the robustness of our meta-analysis, we repeated the analysis following a sensitivity analysis, and we assessed the influence of individual studies on the combined OR by dropping the studies that did not provide an adjusted odds ratio or whose heterogeneity was obvious. A fail-safe number (Nfs) method was used to check for publication bias. P values less than 0.05 were considered significant.
Study retrieval and inclusion
Our search allowed us to find 89 publications. We excluded 63 studies; common exclusions were reports on biochemistry, bacteriology, and immunology. We found a total of 26 retrospective case control studies on CagA-seropositive strains of H. pylori and ischemic diseases eligible for inclusion,5–30 including 11 studies5–15 on CagA status and ischemic stroke, and 15 studies16–30 on CagA status and coronary heart disease. The research population of these studies included patients who were either Chinese or Caucasian. According to this situation, we performed two meta-analyses with studies on CagA status and ischemic stroke and studies on CagA status and coronary heart disease, and also divided the total population into a Chinese subgroup and a Caucasian subgroup, yielding a total of 977 patients in the ‘cases’ and 996 subjects in the ‘control’ groups on CagA status and ischemic stroke; a total of 2157 patients in the ‘cases’ and 2383 subjects in the ‘control’ groups on CagA status and coronary heart disease.
All the included studies adjusted important confounding factors: age, gender, diabetes, hypertension, smoking, social class, BMI, hyperlipidemia, peptic diseases and other pathogenic microorganism infection). Groups were comparable, and most studies performed following a multivariate analysis. All the studies used an enzyme-linked immunosorbent assay (ELISA) or a Western blot to detect seropositivity for the CagA antigen.
Result of the meta-analysis on relationship betweenCagA-seropositive strains and ischemic stroke
The results of test for heterogeneity in the total population and its subgroups were positive in these studies (P=0.39 in Chinese; P=0.77 in Caucasian and P=0.68 in overall population). Therefore a fixed effect model was adopted to combined analysis, and the results of a combined analysis were statistically significant. (OR=2.65, 95% CI: 2.00, 3.52, P <0.00001 in Chinese; OR=2.71, 95% CI: 2.05, 3.59, P <0.00001 in Caucasian; OR=2.68, 95% CI: 2.20, 3.27, P <0.00001 in overall population), suggesting that CagA-seropositive strains may be a determinate predisposing factor for the risk of an ischemic stroke in the total population, moreover, it was also suggested that there was no obvious difference between the results from the Chinese and Caucasian patients. The results of the meta-analysis are shown in Figure 1.
Result of the meta-analysis on relationship betweenCagA- seropositive strains and coronary heart disease
The results of test for heterogeneity suggested there were significant in these studies (P=0.09 in Chinese; P=0.02 in Caucasian and P=0.003 in overall population), we performed a combined analysis using a random effect model, yielding ORs of 2.70 (95% CI: 1.75, 4.18, P <0.00001) in Chinese; 1.92 (95% CI: 1.51, 2.44, P <0.00001) in Caucasian and 2.11 (95% CI: 1.70, 2.62, P <0.00001) in the total population, eliciting that there is a significant trend toward positive association between CagA-seropositive strains of H. pylori and coronary heart disease. There was also no obvious difference between the results from the Chinese and Caucasian patients. The results of meta-analysis are shown in Figure 2.
Sensitivity analysis and publication bias
All the studies retrieved important and potential confounders and classical cardiovascular disease risk factors. However, one study15 had not provided adjusted odds ratio for the relationship between cerebral ischemia and CagA-seropositive strains, and one study's29 heterogeneity was obvious in the subgroup of coronary heart diseases. The sensitivity analysis performed after excluding these studies gave a pooled odds ratio of 2.69 (95% CI: 2.18, 3.31) and 2.18 (95% CI: 1.76, 2.70) respectively. These results were similar to those of a meta-analysis before sensitivity analysis, suggesting that the combined results of the meta-analysis were reliable.
Various fail-safe number methods (with P=0.05 and P=0.01) were applied to evaluate publication bias, Nfs0.05=568.06, Nfs0.01=275.89 with ischemic stroke in the total population; Nfs0.05=620.93, Nfs0.01=300.05 with coronary heart diseases in the total population, which suggested that there was almost no potential for the lack of unpublished data for the negative outcomes in these results of meta-analysis.
In the present meta-analysis we observed moderate association of ischemic stroke, particularly caused by atherothrombosis with CagA-seropositive strains. Moreover, the results showed no obvious difference between Chinese and Caucasian patients. While conducting CagA-seropositive strains as the risk factor of coronary heart disease to combine analysis, we found statistical significance between the CagA-seropositive strains and coronary heart disease in all subgroups and total population. Even by applying sensitivity analysis, no difference was found in the conclusion. This indicated that the conclusion was reliable and credible. To have an internal control, valid in all studies, for this association, we had planned to assess the relationship between CagA- seronegative strains of H. pylori and atherosclerotic diseases. Because there are no specific tests that can be used to detect CagA-seronegative strains, and some studies also did not report the total prevalence of H. pylori infection, we could not calculate the prevalence of CagA-seronegative strains in the case and control groups.
The role of chronic inflammation in atherogenesis has been studied extensively over the past decade. Conceiving atherosclerosis as an inflammatory condition has given rise to a series of hypotheses aimed at finding determinants for the occurrence of cardiovascular and cerebrovascular diseases beyond the traditional risk factors.31 Among these hypotheses, the contributions of chronic infections to the development of atherosclerosis have been investigated in many cross-sectional and prospective studies and are currently regarded as cooperating factors in maintaining a chronic inflammatory status. CagA-seropositive strains infection have been linked to other non-classical markers of atherosclerotic diseases, such as heat shock proteins,4 and the relationship of the infection with these markers is largely unexplored.
The following assumptions could explain our findings. First, CagA-seropositive strains can increase both COX-1 and COX-2 activity in vascular endothelial cells. This increased generation of endothelial cell prostacyclin may play a role in platelet function and inflammatory cell infiltration in the process of an atherogenesis.32 Second, it is well known that CagA-seropositive strains are able to induce an enhanced inflammation in the gastric mucosa.1 Because inflammation plays a major role in atherosclerosis and complicated lesions are associated with an increased inflammatory response,33 the strong inflammatory response related to the infection by CagA-seropositive strains may influence them through an immune-mediated release of cytokines and other substances endowed with proinflammatory properties.14,15
Third, an autoimmune reaction could be postulated. A cross-reactivity between anti-CagA antibodies and vascular wall antigens has been recently demonstrated, suggesting that these antibodies may contribute to the activation of inflammatory cells within atherosclerotic lesions.34 This activation might eventually lead to plaque destabilization. Fourth, it has been suggested that H. pylori, as well as Chlamydia pneumoniae, can directly provoke inflammation within the atherosclerotic plaques. H. pylori DNA has been found in carotid atherosclerotic lesions and has been associated with features of inflammatory cell response.2 Fifth, it has been suggested that H. pylori-induced chronic atrophic gastritis decreases plasma vitamin B12 and folic acid levels, leading ultimately to an increase in circulating homocysteine levels. These substances were confirmed with the atherosclerosis.35
The relationship between infection with virulent strains of H. pylori and carotid intima-media thickness (IMT), a marker of early atherosclerosis, had also been reported. Some studies showed that a strong correlation between infection with CagA-positive strains and IMT. During a 5-year follow-up (from 1995 to 2000), Mayr36 reported the first prospective population-based study demonstrating that infection with CagA-positive but not CagA-negative strains is associated with a larger increase of carotid IMT and significantly increase the risk of carotid atherosclerosis, moreover, the study provided evidence that this association is more pronounced in subjects with an enhanced immune inflammatory response. Diomedi's37 study included 185 consecutive patients with first acute ischemic stroke without control group, demonstrating that infected with CagA-positive strains in atherosclerotic stroke patients is associated with greater IMT and poorer short-term outcome compared with CagA-negative patients. However, Markus's38 cross-sectional study gave contradictory results, which showed H. pylori and the CagA-seropositive strains are not major risk factors for early arteriosclerosis as assessed by carotid IMT after controlling for cardiovascular risk factors (not included age and gender).
Atherosclerotic diseases, especially ischemic stroke and coronary heart diseases are likely to be multifactorial, involving both polygenic and environmental influences. This review supports the notion that atherosclerosis can be regarded as an inflammatory process and that the presence of a chronic systemic inflammatory status is a strong risk factor for the development of coronary artery disease and ischemic stroke.
Possible limitations of our study and the prospects are as follows. First, although a positive association between CagA-seropositives strains and ischemic diseases was found in the review, however, it is important to note that prospective studies on the status of CagA and the risk of atherosclerotic diseases were not included in meta-analysis; these studies were heterogeneous in design, measurement and evaluation which limited combined analysis. Second, although seropositivity to CagA is widely used as a surrogate of infections with toxic H. pylori strains, individual seronegative to CagA may still be infected with virulent H. pylori expressing VacA in culture. Third, the results of the study may not necessarily apply to all populations with a different prevalence of H. pylori infection. Fourth, clinical trials had also addressed the question of a possible positive impact of antibiotic therapy on the clinical burden of cardiovascular diseases.39 More prospective studies of therapeutic trials are still needed to confirm the impact that the eradication of CagA-seropositive strains on the risk of ischemic vascular diseases. Further prospective studies are required, however, to confirm whether chronic CagA-seropositive or seronegative strains of H. pylori infection is an independent risk factor for ischemic diseases caused by an atherosclerotic mechanism.
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