Prevalence of Helicobacter pylori vac A , cag A , dup A and oip A Genotypes in Patients with Gastric Disease

Gastric diseases such as chronic gastritis and gastric cancer are most commonly caused by virulence factors of Helicobacter pylori (H. pylori), such as the vacA, cagA, dupA and oipA genes. Therefore, this study investigated the prevalence and the combination of these virulence factors from patients with gastric diseases. The endoscopic biopsies were obtained from 516 patients with gastric symptoms, 101 of which were from patients with normal gastric tissue, 365 of which were from patients with chronic gastritis, and 50 of which were from patients with gastric cancer. H. pylori and the virulence factors were detected by PCR. The oipA gene exhibited an increased risk for chronic gastritis (p = 0.0296), and the vacA gene demonstrated a risk for gastric cancer from chronic gastritis (p = 0.0002). Based on the combination of the virulence factors, cagA, vacA, dupA and oipA genes exhibited a high prevalence in patients with chronic gastritis and gastric cancer. The cagA+/dupA+ genotype demonstrated a significant correlation in patients with normal gastric mucosa (p = 0.0278). In the chronic gastritis group, a significant association was observed between the cagA+ and the vacA s1m1 genotypes (p < 0.0001), the cagA+/dupA+ genotypes (p = 0.0183), the dupA+/oipA+ genotypes (p < 0.0001), and the dupA+/vacA s1m1 genotypes (p = 0.0008) genotypes. This study revealed a high prevalence of the combination of cagA, vacA, dupA, and oipA genes, which contributed to the risk of developing gastroduodenal diseases. Furthermore, this is the first study to reveal a high prevalence of the oipA gene in H. pylori isolates in Brazil.


Introduction
Helicobacter pylori (H.pylori) is a gram-negative and microaerophilic bacterium that infects approximately 50% of the world's population.However, the prevalence of the infection varies between developed and developing countries.A higher prevalence of H. pylori infection (75% -83%) was found in Latin American countries than in Japan (40%) and other developed countries [1] [2].
H. pylori infection is the main cause of peptic or duodenal ulcers, gastritis, and gastric cancer.The bacterium colonizes the gastric mucosa by using urease activity to resist the hostile acidic condition of the stomach.After surviving in the stomach environment, flagellar motility is necessary to move the bacterium through the gastric epithelium cells.The interaction of H. pylori adhesins and cellular receptors supports gastric mucosa infection, and it is followed by the secretion of virulence factors (such as vacA, cagA, dupA and oipA genes) which cause tissue damage [1] [3] [4].
The vacA gene encodes the vacuolating cytotoxin A, which may participate in several cellular activities, including the inhibition of T-cell proliferation and activation and the onset of proinflammatory response.These activities depend on differences in the gene's structure, the signal region, or s (s1 and s2), and the middle region, or m (m1 and m2).
cagA (cytotoxin-associated gene A) is the most frequently studied virulence factor encoded by the cag pathogenicity island (PAI).This protein is injected into gastric cells, causing oncogenic activity and modification to normal cell differentiation, such as cell polarity and cell adhesion.This gene is associated with increased inflammatory response and risk of gastric cancer and peptic ulcers [7] [8].
The dupA (duodenal ulcer promoter gene A) gene was identified as the first disease-specific virulence factor in 2005, for its ability to increase the risk for duodenal ulcers.It was also reported that dupA had a suppressive effect on gastric cancer.Recent studies have reported an association between the dupA gene and elevated IL-8 production from gastric epithelial cells in vivo.However, the presence of dupA and the increased IL-8 production in vitro is still under discussion [6] [9].
oipA (outer inflammatory protein A) was identified in 2000 by Yamaoka, et al. [10] as one of the outer membrane proteins.The oipA gene has two functional statuses: "on", which reveals the functional gene; and "off", which reveals the non-functional gene.This condition is regulated by a repair mechanism known as slipped strand mispairing, which is based on the number of CT dinucleotide repeats in the 5' region of the oipA gene [7] [8].The presence of oipA and cagA and/or cag PAI seems to be associated with β-catenin signaling, which promotes the opening of cell-cell junctions [6].Furthermore, the oipA's "on" status is associated with higher risks of gastric cancer and peptic ulcers than it is with gastritis and functional dyspepsia [11].
These virulence factors may be related by acting synergistically with each other, thus increasing the risks of several gastric diseases [6].Therefore, more studies on the clinical importance of H. pylori virulence markers are necessary, particularly research that considers the recent dupA and oipA genes, the functions of which have not yet been clarified.
Considering the unclear epidemiological aspects of H. pylori, the aim of present study was to detect status of cagA, vacA, oipA and dupA genotypes in H. pylori isolated from patients with chronic gastritis and gastric cancer.

Study Populations
Five hundred and sixteen gastric mucosa biopsy samples were collected from patients with gastric symptoms who had undergone an endoscopic investigation at the Bauru State Hospital (HEB), the Gastrointerology Department of the Marilia School of Medicine (FAMEMA), and the Federal University of São Paulo (UNIFESP).Informed consent was obtained from all patients, and the study was approved by the Research Ethics Committee of Universidade do Sagrado Coração (USC) in Bauru, São Paulo, Brazil (No. 1.045.181).

DNA Extraction and H. pylori Detection
Genomic DNA from the gastric biopsy samples was extracted using the QIA amp ® tissue kit (Qiagen, Germany) according to the manufacturer's instructions.Polymerase chain reaction (PCR) was used to detect the presence of Helicobacter pylori and virulence factors from the vacA, cagA, dupA and oipA genes.All samples were submitted to diagnosis of H. pylori but just the positive samples were submitted to detection of virulence factors.PCR and the primer information from each amplicon are summarized in Table 1.

Statistical Analysis
To estimate the statistical differences between the diseases and the various genotypes, the Chi square test and Fischer's exact test were used.The risk was estimated using an odds ratio (OR) and a 95% confidence interval (CI).A p value of 0.05 was considered statistically significant.

Results
Helicobacter pylori was detected in 256/516 (49.6%) of the samples.The prevalence of H. pylori and its virulence factors in each group of patients (the normal group, the chronic gastritis group, and the gastric cancer group) are detailed in Table 2, as are the ORs, the 95% CI, and the p value.
The results suggest a significant association between gastric diseases and the presence of H. pylori.Likewise, the oipA gene was found to be associated with an increased risk of chronic gastritis.In the comparison between the groups of patients with chronic gastritis and the group with gastric cancer, the OR (2.46), the 95% CI (1.244-4.861),and the p value (0.0089) of the bacterium's presence also indicated a significant association.

After the detection of the virulence factors in this comparison (Gastritis and Gastric
Cancer groups), only the vacA gene was found to be statistically significant (OR: 7.314; 95% CI: 2.157-24.8;p = 0.0002).
Based on the combination of the cagA gene with the other virulence factors represented in Table 3, a significant correlation was observed between the cagA and the dupA gene in the normal group (OR: 24.2; 95% CI: 0.9298-629.8;p = 0.0278).In the   chronic gastritis group, two associations were found to be statistically significant.The first relationship was between the cagA and the vacA s1m1 genes (OR: 16.02; 95% CI: 7.364-34.85;p < 0.0001), and the second was between the cagA and the dupA genes (OR: 1.962; 95% CI: 1.126-3.419;p = 0.0183).However, no significant correlations werefound between the virulence factors in the gastric cancer group.

Discussion
The association between Helicobacter pylori and the development of duodenal and peptic ulcers, chronic gastritis, and an increased risk for gastric cancer is now a consensus [12].Thus, our results demonstrate a significant relationship between the presence of H. pylori and the increased risks for gastric diseases.The results are in accordance with many studies that have been published since the discovery of H. pylori [12] [13] [14] [15] [16].These clinical outcomes vary according to the host and H. pylori factors, such as the vacA, cagA, dupA, and oipA genes.
VacA activity depends on the structure of the gene.Studies have found that in Western countries, strains containing the s1m1 genotype are associated with a higher risk of gastric disease, as well as with enhanced proinflammatory response [17].Similar to our results, Asrat, et al. [18] and Suzuki, et al. [19] also found s1m1 to be the most common vacA genotype.Interestingly, all of these virulence factors have a mutual relationship; for example, cagA-positive strains often have the s1m1 genotype, and several studies have provided evidence of an association between the cagA+/vacA s1m1 genotype and the risk of gastric disease, which was also found in our study [20] [21] [22].However, the significant association between this genotype and gastric diseases was only observed in the chronic gastritis group (p < 0.0001).No associations were found in the gastric cancer group, a result which conflicts with the data provided by Suzuki, et al. [21].
When the oncoprotein cagA is present in host cells, the risks for peptic ulcer and gastric cancer have been found to increase in Western countries when compared to cagA-negative strains [16] [19].This data was found to be inconsistent with our study, because the cagA gene alone was not found to be associated with any clinical outcome; however, our report is consistent with the results found by Kim, et al. [23].
The prevalence of the cagA gene may vary according to the region.In Eastern countries, cagA-positive strains reach almost 100%, while in some Western countries the prevalence is less than 50% [6].The present study detected 50% or fewer cagA-positive strains among a cohort of Brazilian patients.This number is similar to what Sasaki, et al. [24] observed in Ecuador (45.9%) and Panama (20%), though it is not in agreement with what Yamaoka, et al. [25] found in Japan or what Watada, et al. [22] found in Colombia.
In our report, the cagA gene also exhibited a significant association with another virulence factor, the dupA gene, which is a protecting factor against gastric cancer and which is associated with an increased risk for duodenal ulcer [26] Gomes, et al. [27] also found a correlation between cagA-positive and dupA-positive strains, but the association was in patients with duodenal ulcers.In a meta-analysis, Shiota, et al. [26] reported a higher prevalence of dupA in Western countries than in Eastern countries.The study performed by Gomes, et al. [27] revealed an increased prevalence of dupA in Brazilian patients with chronic gastritis (92.3%) and gastric cancer (87.6%), a finding which differed from that provided by Lu et al. (2005) in a study on strains from Colombia.It also differs from our findings.This difference likely occurred because the pa-tients were from different regions in Brazil.
One interesting point about the dupA gene was that we observed a statistically significant association between it and the vacA s1m1 genotype.This fact has not been described previously.The only report on this association was provided by Arachchi et al. (2007), who observed a correlation between s1 and m1 alone.Interestingly, the dupA gene also exhibited a significant association with the oipA gene in our study; both genes are involved in the induction of IL-8 secretion [7].However, our findings differ from those of Souod, et al. [28], who reported a statistically significant association with the dupA+/oipAgenotype, and with the same p value as ours (p < 0.0001).Though the clinical outcomes resulting from the presence of the oipA gene are still unclear, Souod, et al. [28] similarly observed a significant correlation between the oipA gene and chronic gastritis.In addition, they found no relationship with gastric cancer, as Yamaoka, et al. [29] also reported.

Conclusion
Taken together, our results suggest that the high prevalence of virulent factors contributes to the risk of developing gastroduodenal diseases.We hypothesize that the more virulent combination may be a trigger for chronic gastritis, a precursor lesion of gastric cancer.This is the first study to reveal a high prevalence of the oipA gene in H. pylori isolates in Brazil.Furthermore, this study reveals a high prevalence of the combination of cagA, vacA, dupA, and oipA genes.Our findings reinforce the importance of understanding the correlation between the virulence of H. pylori and host susceptibility.

Table 2 .
Distribution of the Helicobacter pylori genotypes and the risk of developing chronic gastritis and gastric cancer Comparisons between the normal group and the chronic gastritis group and the normal group versus the gastric cancer group.

Table 3 .
Association between the cagA gene and the vacA, dupA, and oipA genes.
n: number of samples.*association statistically significant.