Genetic Analysis of cagA and vacA Genes in Helicobacter Pylori Isolates and Their Relationship with Gastroduodenal Diseases in the West of Iran

Background Helicobacter pylori have different virulence factors which are associated with several gastroduodenal diseases; however, this association is variable in different geographical regions. Data of genotypes of Iranian H. pylori isolates are few. Objectives The aim of the current study was to investigate the cagA/vacA genotypes of Helicobacter pylori isolates and determine the relationship between these genotypes with respect to different gastric disorders in patients of Chaharmahalo Bakhtiarian. Materials and Methods In this cross-sectional study, gastric biopsies were taken from 200 patients with gastrodoudenal diseases. Histopathological features were recognized by specialist. The samples were subjected to PCR for detection and identification of ureC, cagA and vacA genes. Results The frequency of the vacA genotypes, sa1/m1, s1a/m1b, s1a/m2, s1b/m1a, s1b/m1b, s1b/m2, s1c/m1a, s1c/m1b, s1c/m2, s2/m1a, s2/m1b and s2/m2 were 27(6.6%), 8(4.3%), 45(28.04%), 7(3.7%), 5(2.5%), 10 (6.1%), 12 (7.4%), 4 (2.5%), 18(11%), 6(3.7%), 0 and 22(13.5%) respectively. The cagA gene was detected in 92% of strains. Based on our findings, it seemed that cagPAI and vacA s1 genotypes were associated with some gastric disorders in patients with H. pylori. In this region, the isolates carrying s1a/m2 were the most prevalent. Conclusions We found considerable relationship between s1a/m1a, s1a/m2, s2/m2 and s1c/m1a and some gastric disorders. Further studies about the role of H. pylori virulence factors and gastric disorders were recommended.


Background
Helicobacter pylori is major causes of chronic gastritis. They are also involved in the pathogenesis of several diseases including gastric and duodenal ulcers, gastric adenocarcinoma and mucosa-associated lymphoid tissue lymphoma (1). Several H. pylori virulent genes have been identified to contribute to the severity of these diseases.
The pathogenicity island (PAI) is the most important virulent factor of the bacterium that encodes a type IV secretion apparatus. The cagA gene is located at the end of the cag PAI (2)(3)(4)(5). Another important virulence factor of H. pylori is a vacuolating cytotoxin (VacA), which is associated with injury of epithelial cells. The vacA gene is present in nearly all strains of H. pylori but it is poly-morphic, comprising variable signal regions (type s1 or s2) and mid-regions (type m1 or m 2). The type s1/m1 vacA strains causes more epithelial cell damage than type s1/ m2, whereas type s2/m2 and the rare s2/m1 are non-toxic due to the presence of a short 12-residue hydrophilic extension on the s2 form (3,6). The s-region is classified into s1 and s2 types and the m-region into m1 and m2 types. The s1 type is further classified into s1a, s1b and s1c subtypes, and the m1 into m1a and m1b subtypes. The mosaic combination of s and m-region allelic types determines the particular cytotoxic and, consequently, the pathogenicity of the bacterium (7,8). There is a geographical variation in the vacA genotypes (9,10). For example, studies have consistently shown that vacA s1a strains predominate in northern Europe, s1b in Central and South America, Spain and Portugal, s1a and s1b in the USA and s1c in East Asia (10). These differentiations may cause variations in the prevalence of gastric diseases in these areas. Thus, existing data are contradictory and cannot explain the pathogenic role of H.pylori in the development of different gastric diseases. Furthermore, it might be useful to know the genetic diversity of H.pylori strains in Chaharmahalo Bakhtiari, one of the risky provinces of Iran.

Objectives
The aim of the current study was to investigate the cagA/vacA genotypes of Helicobacter pylori isolates and determine the association between these genotypes with different gastric disorders in Chaharmahalo Bakhtiarian patients.

Collection of Patient Samples
From June to November 2009, 200 consecutive patients with dyspeptic symptoms attending the endoscopy suite of gastroenterology section of Hospital of Shahrekord University of Medical Sciences (SUMS) enrolled in the study. The questionnaires, including medical history and demographic data, were recorded for each patient. All studied patients signed an informed consent form before endoscopy and declared their willingness to allow the application of their anonymous data for research purposes. For each patient, two biopsy specimens were taken from the antrum using a disinfected endoscope. One piece of each specimen was examined by Rapid Urease Test (RUT) for detection of H. pylori. RUT was performed with a Gastro urease kit (Baharafshan, Iran). The second piece from positive samples in RUT was placed in 0.1 ml of sterile saline solution and was sent to Biotechnology Research Center of Islamic Azad University, Shahrekord Branch for further studies.

Statistical Analysis
The data were analyzed by SPSS software (Version 17.spss Inc, USA) and P value was calculated using Chi-square and Fisher's exact tests to find the significant relationship. P value less than 0.05 was statistically significant.

Discussion
H. pylori are one of the most genetically diverse bacterial species which may be involved in the complex variety of gastro duodenal diseases in infected patients all over the world (13)(14)(15)(16). The geographic prevalence of distinct H. pylori genotypes remains largely unknown (15). For instance, in Japan, South America, Turkey and Pakistan, the prevalence is more than 80%, while in Scandinavia and England, the prevalence is between 20% and 40% (3). The prevalence of this bacterium in Iran is 60-90%, indicating that Iran is a highly risky region for H. pylori infection. The prevalence of this bacterium was 82% in our study indicating that our findings are consistent with previous reports in Iran (16)(17)(18). The vacA genotypes show considerable variability in different geographic regions (3). According to our results, 80% of samples had vacA s1a, b, c, also m2 genotypes and s1a/m2 was predominant in H. pylori isolates. This finding is somewhat similar to Europe and North America, where vacA s1a, s1b and m2 are predominating too. Our isolates were similar to those isolated from East Asian isolates where s1c is predominant (15). This study showed that Iranian H. pylori isolates are very diverse in genotype and contain the East and the West elements. H. pylori strains concluding cagA gene are more virulent than cagA-negative strains (3). The prevalence of cagA-positive H. pylori varied from one geographic region to another, e.g., 97% in Korea, 94% in Malaysia, 90% in China, 78% in Turkey and 53% in Kuwait (19). In this study, we found cagA gene in 92% of the H. pylori-positive population. This finding did not demonstrate the role of cagA as predictive marker for increased virulence feature of H. pylori, because of the high positivity of this gene in all H. pylori isolates. A strong association between the cagA and vacA status and peptic ulcer disease has been reported (20)(21)(22). Beil et al. suggested that the increased inhibitory effect of cagA-positive, cytotoxin-producing strains on mucin synthesis could be considered as a possible mechanism which is responsible for the increased risk of developing peptic ulceration with these H. pylori strains (9). Gzyl et al. found that cagA gene correlated with active gastritis in infected children and adults. They also found that the majority of H. pylori strains carrying s1/m2 vacA alleles were responsible for the higher levels of cytotoxin production (10). Our data were in agreement with those of Gzyl et al. and Beil et al., which suggest that H. pylori strains with cagA and vacA s1 genotypes are associated with more severe gastritis (9, 10). Investigator's opinions about the association between vacA genotypes and gastric disorders were different. For example, in Iran, Jafari et al. found no correlation between them, (3) whereas Mohammadi et al. and Molaei et al. found that s1a allele were associated with more severe inflammation (15,23). As results showed, we found an association between some diseases and some vacA genotypes but we couldn't introduce any allele as a marker of a disease. For instance, s2m2 strains that are non-toxigenic in most regions of the world and are associated with NUD diseases were surprisingly more prevalent in PUD than in NUD patients and had direct association with duodenal ulcer in our evaluation. In Thailand, Japan, Korea, Colombia and America, no association had been found (21) whereas in Cuba, Lebanon, Hung Kung, China and most of the European countries, a significant association between s1 allele and PUD diseases had been reported (20,21,24,25). Data analysis revealed a significant association between Duodenal ulcer and s1a/m1a (P = 0.04), s1a/m2 (P = 0.02) and surprisingly s2m2 (P = 0.05) genotypes. We observed an association between Gastritis diseases and s1c/m2 (P = 0.01). We didn't find any relationship between other diseases with regard to vacA genotypes. In conclusion, we found that the cagA-positive s1/m2 H. pylori were dominant genotypes in the patients under study. The cagA gene positivity rate was probably not closely associated with severity of the disease. H. pylori strains including vacA s1 genotype were associated with more severe gastritis. laboratory experiments; Ranjbar R and Souod N analyzed the data, interpreted the results and wrote the paper.

Financial Disclosure
All authors declare that there is no financial disclosure.

Funding Support
The study was supported by Young researcher's club financially.