Prevalence of Helicobacter pylori cagA and sabA Genotypes in Patients with Gastric Disease

Gastric cancer is one of the most common types of cancer worldwide. Helicobacter pylori is considered one of the most important causes of this condition specially because of its virulence markers as sabA and cagA. Therefore, we aim to investigate the relation between these markers and the gastric diseases in 400 patients who underwent upper digestive endoscopy. To detect the bacteria and its genes by Polymerase Chain Reaction (PCR), the presence of H. pylori was significant when comparing the groups control vs. cancer (p value < 0.0001) OR [95% CI] 12.73 (5.45 29.69) and the groups control vs. chronic gastritis (p value < 0.0001) OR [95% CI] 12.99 (7.44 22.66). cagA was statistically significant considering its presence when comparing the chronic gastritis vs. cancer groups (p value = 0.0434) OR [95% CI] 2.44 (1.021 5.845). Associating both sabA and cagA, we found a statistically significant result (p value < 0.0001) OR [95% CI] 13.68 (3.95 47.33) considering the gastritis vs. cancer groups. Helicobacter pylori is directly associated to gastric diseases such as gastritis and cancer and its virulence markers: sabA and cagA increase the injury process to the gastric epithelium making the host more susceptible to cancer.


Introduction
Gastric cancer is one of the most common types of cancer worldwide and the third leading cause of cancer death.It is responsible for about 720,000 deaths per year around the world, and its occurrence is very frequently in Asian countries [1] [2].The onset of cancer usually starts with lesions, which evolve into an in-flammation that tends to be followed by chronic gastritis, gastric atrophy, and finally gastric cancer [3].Its progression is related to genetic characteristics of the host, environmental factors, and the most important parameter: the presence of Helicobacter pylori infection as well as its genotype [4].
H. pylori is a Gram negative, microaerophilic bacterium that colonizes half of the world's population and whose niche is the stomach [4] [5].Its infection is highly successful due to the interaction of this bacterium with the gastric epithelial cells, and for that reason; it is considered one of the most important causes of gastric cancer [6] [7] [8].
The sialic acid-binding adhesin (sabA) is an outer membrane protein that plays an important role in the first contact of H. pylori with the gastric tissue [9].
This marker determinates the initial and permanent colonization of the bacteria and directly influences the extent of damages by this pathogen.sabA acts in adhesion of Helicobacter pylori by binding to a specific receptor present in human stomach named sialyl-dimeric-Lewis x (Le x ) [10] [11] [12].This binding characterizes a successful and more aggressive infection making the intense inflammation permanent in which susceptive lesions can eventually evolve into gastric cancer [13].
Another virulence marker of H. pylori is the cytotoxin-associated gene A known as cagA, which is related to gastric inflammation [12].The genome of H. pylori includes a component called cag-PAI island, which encodes the type IV secretion system.This system perforates the cells of the gastric epithelium, allowing the bacteria to deposit in the host molecules, such as the cagA protein, which regulates the metabolism of these cells [14].This marker is considered one of the most virulent because its presence is usually associated with the etiology of peptic ulcers and gastric cancer [10].
Considering the high occurrence of H. pylori, the present study aimed to investigate the relation of its genes sabA and cagA with gastric diseases and gastric cancer.

Statistical Analysis
Statistical analysis was performed using the two-tailed Chi-square test with Yates' correction and/or Fischer's exact test.Differences were considered significant when p value was less than 0.05.All statistical analyses were performed with software GraphPad Prism 5.0.

Results
In the 400 analyzed gastric mucosa samples, Helicobacter pylori was detected in 222 (55.5%) (Table 1).The result was significant for the presence of H. pylori when compared the control vs. cancer groups (p < 0.0001) OR (95% CI) 12.73 (5.45 -29.69) and the control vs. chronic gastritis groups (p < 0.0001) OR (95% CI) 12.99 (7.44 -22.66).These results found a significant relation between H. pylori and gastric diseases considering the effects of this bacterium in gastric mucosa.
The cagA gene was detected in 102 (45.94%) H. pylori positive samples also divided in the control, chronic gastritis, and cancer groups, with 8, 86, and 8 samples, respectively (Table 1).This virulence marker was statistically significant considering its presence when comparing the chronic gastritis vs. cancer groups (p = 0.0434) OR (95% CI) 2.44 (1.021 -5.84).Table 1 showed the results of H. pylori and virulence markers in each group of patients.

Associations between cagA and sabA
Analysis of the association between cagA and sabA found no significant results (Table 2).However, the combination of both markers with the groups of patients produced a statistically significant result (p < 0.0001) OR (95% CI) 13.68 (3.95 -47.33), considering the gastritis vs. cancer groups (Table 3).This result suggests that when these markers act together they potentiate the action of H. pylori by increasing the inflammatory process that can evolve from chronic ga- stritis into cancer.Table 3. Distribution of the combination between cagA, and sabA genotypes and the risk of gastric disease.Comparisons among: control vs chronic gastritis (a) ; control vs gastric cancer (b) ; and chronic gastritis vs gastric cancer group (c) .

Discussion
Our results show that Helicobacter pylori is significantly related to gastric diseases, such as gastritis, and increase the risk of gastric cancer.Considering that this bacterium is capable of establishing an intense inflammatory process in the human gastric epithelium, these results were expected, because since the discovery of H. pylori, other works have concluded the sam [22] [23] [24] [25].
The outer membrane protein sabA, which interacts with a specific receptor present in gastric epithelium, can be responsible for facilitating binding between the bacteria and the host [26].This gene has been identified in more than a half of the H. pylori positive studied population; however, no statistically significant results were found in this work.Our results are in the line with those published in Pakbaz et al. (2013) [13], but not in agreement with Oleastro et al. (2013) [27] who found association between this adhesin and gastric cancer in western population.
Although we did not find any significant results for the sabA gene, it is still recognized as a virulence marker for its ability to promote intense recruitment of neutrophils and establishment of a persistent colonization [28] [29].
One of the most aggressive virulence markers expressed for Helicobacter pylori and probably the most studied is cagA [4] [10].This gene confers to the bacteria the ability to modulate the cell metabolism of the host [30], and its presence is related to development of ulcer and gastric cancer [31].Our data indicate that cagA gene is a risk factor for the onset of gastric cancer in patients who have previously injured gastric epithelium.This result is similar to the results found by Yamaoka et al. (2006) [32] and Oldani et al. (2009) [31].
It is known that H. pylori depends on a strong initial bond to the gastric tissue to conclude a successful and permanent infection, and it has developed mechanisms that greatly help this process [27].Once the infection is established, H. pylori starts to release its toxins that regulate the host cells and assist in its adaption to the stomach niche [12] [33].
Both sabA and cagA are important virulence markers for Helicobacter pylori [11] as they participate in the processes mentioned above.Our results indicate that these genes taken together make the infection process more aggressive and increase the lesion of the gastric epithelium making the host more susceptible to cancer.Our data about this association is consistent with the data presented by Backert et al. (2011)  [33].
We were expecting to have positive results correlating sabA only and the gastric cancer.We believe that we haven't got this result because of the sample size and the target population of this work.addition, we had difficulty finding in the literature works that approached the questions of this article in a similar way to ours and, therefore, there were few comparisons of results that we were able to accomplish.

Conclusion
Taken together, our data confirm that Helicobacter pylori is directly related to the emergence and evolution of gastric diseases, especially chronic gastritis and cancer.Its virulence markers sabA and cagA are responsible for a persistent and very aggressive infection that moves the patient's situation from an initial injury of the gastric epithelium to a greater susceptibility for gastric cancer.

Table 1 .
Distribution of the Helicobacter pylori, sabA and cagA genotypes, and the risk of developing chronic gastritis and gastric cancer.
*Results statistically significant when compared control vs. cancer group (p< 0.0001) and control vs. chronic gastritis group (p < 0.0001).▲ Results statistically significant when compared chronic gastritis group vs. cancer group (p = 0.0434).

Table 2 .
Association between the cagA and sabA genes in the studded groups.