Helicobacter Pylori and CagA : Relationships With Esophageal and Gastroduodenal Disorders in Iranian Patients

1Department of Microbiology, School of Medicine, Babol University of Medical Sciences, Babol, IR Iran 2Department of Bacteriology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, IR Iran 3Department of Laboratory Medical Science, Faculty of Paramedicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, IR Iran 4Baqiyatallah Research Center for Gastroenterology and Liver Disease, Baqiyatallah University of Medical Sciences, Tehran, IR Iran


Background
After discovering the H. pylori pathogenesis by Barry and Marshal, many research groups have focused on the H. pylori virulence factors and co-relation between its virulence factors and pathological or clinical outcomes (1)(2)(3).
It has been proved that the bacterium is the main cause of gastritis, gastric and duodenal ulcer and gastric cancer (4,5).WHO has declared that H. pylori should be recognized as type I carcinogen (6).
The pathogenesis of H. pylori is complicated, and many infected people are asymptomatic (7,8).Moreover, H. pylori virulence factors play an important role in clinical outcomes, and CagA is the most important virulence factor of the bacteria (9).Additionally, it has been proved that 60% of H. pylori expresses this 128 kDa protein, and many investigators have reported that there is a corelation between H. pylori CagA positive and severity of stomach disorder (2).These strains are leading cause of IL-8 production, actins rearrangement, cell cycle interference, inducing of oncogenes expression and eventually damage to cells (10)(11)(12).Some research groups have reported that H. pylori can interfere to signal transduction and cell cycle via CagA.Thus, injection of this virulence factor into host cells causes activation of NF-kB and turning cells into cancer (13).The rate of H. pylori carriers in Iran is very high, and there are increasing reports of gastric cancer and stomach disorder in this region as well (14,15).

Objectives
The main aim of present study was to assess a significant relationship between presence of CagA and severity of clinical manifestations in esophageal and gastroduodenal disorders.

Patients
The present study was conducted from March 2012 to August 2013.A total of 240 gastric biopsies were collected from patients who were referred to general hospitals in Tehran (Iran).

Biopsy Sampling
Three sets of biopsy specimens were obtained from the antrum, and each set of three specimens was divided as follows: One each for rapid urease tests; one for histological examination; One for Polymerase Chain Reaction (PCR) assay.

Rapid-Urease Test
Antrum biopsy specimens were put into a semisolid 2% urea agar and the results were observed 0.5 to 4 hours incubation at room temperature.

Histological Examination
The specimens were fixed by formalin, embedded in paraffin and stained by H and E to find out the severity of gastritis.

Preparation of Samples for Polymerase Chain Reaction
Genomic DNAs were extracted from gastric biopsy specimens using the extraction kit (Sinagen, Iran) according to the company instructions.The presence of H. pylori DNA in biopsy samples were detected by PCR amplification of H. pylori ureC gene (Table 1).In addition, cagA gene was detected by PCR amplification of a 297-bp region in the cagA gene as previously described (Table 1) (16,17).H. pylori ATCC49503 was used as positive control.

Statistical Analysis
The SPSS software (Chicago, Ill., USA) was used for data analysis and for discovering a significant different between the groups we used the chi-square test.A P < 0.05% was accepted as statistically significant.

Results
The rapid-urease test was positive for 108 (45%) of biopsy specimens and the color change of semi-solid media indicated that the specimens had unease activity of H. pylori.

PCR Based ureC Gene
The presence of H. pylori was detected in 45% (n = 108) biopsy samples.The ureC (glmM) gene was amplified in all of urease positive samples 35.5% (n = 108) and PCR method did not miss any urease positive biopsy specimen.

Endoscopic Findings
Demographic information and endoscopic finding were shown in Table 2.According to the endoscopic findings the rate of lesion and duodenum inflammation was more in patients who carried CagA positive H. pylori strains (P ˂ 0.05).Whereas there is less inflammation and sever lesions in esophagus were found in CagA negative H. pylori strains (Table 2).

Discussion
Our study demonstrates a strong relationship between CagA and esophageal and gastroduodenal disorders in Iran.Moreover, our investigation has interestingly revealed that the number of CagA negative in females is considerably larger than CagA positive H. pylori, while there was no statistically significant difference between CagA positive and negative among males.
Although presence of H. pylori in stomach may result in gastritis and gastric cancer, all infected individuals don't express clinical manifestations.Therefore, a number of people remain as healthy carriers (18).There are several factors which contribute to emerge clinical outcomes including bacterial virulence factors, host genetics and unhealthy lifestyle (19).
The bacterial virulence factors play a main role in the pathogenesis of H. pylori.Furthermore, there is much concrete evidence which H. pylori virulence factors are essential to express clinical symptoms (20).In addition, it has been proved that CagA is the most important virulence factor in the bacteria, and individuals infected by CagA positive H. pylori are at high risk of developing gastric adenocarcinoma and duodenal ulcer disease (21).
Even though H. pylori contributes to gastro-duodenal diseases, there are a number of reports that the bacterium may play a protective role against esophageal adenocarsinoma (22).Moreover, some investigators have claimed that CagA positive H. pylori have an inverse association with esophageal adenocarcinoma (23).The present study has revealed that there was a significant difference between CagA positive and negative H. pylori in patients with some esophageal disorders.To further expand, while we did not find significant difference between CagA positive and negative in esophagitis and esophagus lesion grade B, we discovered that the number of CagA negative H. pylori was larger than CagA positive in esophagus lesion grade A, C, and D (P ˃ 0.05).Therefore, our results have shown that cagA may have a protective effect on some esophageal diseases.
However, CagA positive H. pylori presents in 60 -70% all isolated strains, and some epidemiological studies have shown that this virulence factor contributes to developing gastritis, peptic ulcer and gastric cancer (24,25).Our finding confirmed the previous investigations which are performed in Iran (26,27).Although, there was no significant difference between CagA positive and negative H. pylori in gastric mucosa atrophy and deformation of duodenum, we have figured out that the number of CagA positives was larger than CagA negative H. pylori in gastric antrum and duodenum ulcer.Our results showed that the percentage of CagA positive H. pylori is lower than other countries.
In conclusion, our findings have confirmed a number of previous reports which had claimed that CagA positive H. pylori probably benefits for preventing from esophageal disorders and this virulence factor has protective effects by unclear mechanism.On the contrary, our results have shown that CagA positive H. pylori was leading cause of gastric and duodenal ulcers.Moreover, while there was no significant difference between CagA positive and negative in men we have found that the number of CagA negatives was remarkably larger than CagA positives in women.

Figure 1 . 5 E
Figure 1.Prevalence of cagA H. pylori in Different Esophageal and Gastroduodenal, a P < 0.05% Was Accepted as Statistically Significant