Distinct virulence factors of Helicobacter pylori have been associated with clinical outcomes of the infection; however, considerable variations have been reported from different geographic regions. Data on genotypes of Iranian H. pylori isolates are sparse. The aim of this study was to detect cagA/vacA genotypes, and to determine correlations among different cagA/vacA genotypes and gastrodoudenal diseases in Chaharmahal and Bakhtiarian patients for the first time.
Methods: In this cross-sectional study, gastric biopsy was taken from 200 patients with gastrodoudenal diseases. The specimens were processed for DNA extraction and identification of glmM gene. The vacA subtypes and cagA gene were tested by PCR. Histopathological features were recognized by endoscopist.
Results: The frequency of the vacA alleles, 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 respectively 27(6.6%), 8(4.3%), 45(28.04%), 7(3.7%), 5(2.5%), 18(11%), 6(3.7%), 0 and 22(13.5%). In this region s1a/m2 strains were the most prevalent strain and there was a considerable relationship between s1a/m1a, s1a/m2, s2/m2 and s1c/m1a and some gastric disorders.
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Conclusion: Based on our findings, it seems that cagPAI and vacA s1 genotypes are associated with some gastric disorders in patients infected by H. pylori. Further studies about the role of H.pylori virulence factors and gastric disorders are recommended.
Keywords: Helicobacter pylori, vacA, cagA, Gastroduodenal diseases.
Helicobacter pylori is a major cause of chronic gastritis and involved in the pathogenesis of several diseases such as gastric and duodenal ulcer, gastric adenocarcinoma and mucosa-associated lymphoid tissue lymphoma. . Several H. pylori virulent genes have been identified to contribute to the risk and severity of these diseases. These include the cag pathogenicity island (PAI) that encodes a type IV secretion apparatus. [2,3]. The cagA gene is located in the end of the cag PAI and has been proposed as a marker for the cag PAI and the presence of the cagA gene or cagA special type (eg, cagA1a in East Asian strain) is associated with more severe clinical outcomes than the absence of the gene. [4,5].
Another important virulence factor of H. pylori is a vacuolating cytotoxin (VacA), which is associated with injury to epithelial cells. The vacA gene is virtually present in all H. pylori and has at least two variable parts, the signal or s-region, and the middle or m-region. . The s-region is classified into s1 and s2 types and the m-region into m1 and m2 types. The s1 type is further sub typed into s1a, s1b and s1c subtypes and the m1 into m1a, m1b and m1c subtypes. [7,8]. The mosaic combination of s- and m-region allelic types determines the production of the cytotoxin and is, thereby, associated with pathogenicity of the bacterium. vacA s1m1 strains produce a large quantity of toxins, s1m2 strains produce moderate quantities and s2m2 strains produce very little or no toxins. . vacA s1a strains appear to be more pathogenic than s1b or s2 strains and are found more frequently in patients with ulcer diseases, [9,10] although this is not consistent. [11,12]. vacA m1 strains are associated with greater gastric epithelial damage than m2 strains. . There is geographic variation in the vacA genotypes. [7,13]. For example, studies have consistently shown that vacA s1a strains predominates in northern Europe, s1b in Central and South America, Spain and Portugal, s1a and s1b in the USA and s1c in East Asia. . These variations may contribute to the varying prevalence of gastric diseases in these areas. Thus, existing data is contradictory and cannot explain the pathogenic role of H.pylori in the development of different gastric diseases. Consequently, it might be useful to know the genetic diversity of H.pylori strains in Chaharmahal and Bakhtiari province of Iran, a region with a high frequency of H.pylori-induced gastric disease.
The aim of this study was to detect cagA gene and vacA subtypes of H.pylori strains isolated from patients with gastroduodenal diseases and then determined local dominant cagA/vacA genotypes of the organism.
Materials and Methods
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Totally 200 consecutive patients with dyspeptic symptoms attending the endoscopy suite of gastroenterology section of hospital of shahrekord university of medical sciences from June to November 2009 were enrolled. Each patient's history sheet was examined in detail and findings were recorded on standard Performa including demographic data. All patients read and signed an 'informed consent' form at the beginning of endoscopy and declared their willingnesses for the application of their anonymous data for research purpose. 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. Rapid urease test was performed with a Gastro urease kit (bahar-afshan, Iran). A second piece from positive samples in RUT was placed in 0.1 ml of sterile saline solution and sent to biotechnology research center of Islamic Azad University, Shahrekord Branch.
DNA was isolated from biopsy specimens using Genomic DNA purification kit (DNPTM, CinnaGen, Iran) according to manufacture recommendations. Primers sequences used for PCR detection of H. pylori were as follows: ET-2U (5'-CCC TCA CGC CAT CAG TCCCAA AAA-3') and ET-2L (5'-AAG AAG TCA AAA ACG CCC CAA AAC-3'). . Primers used for PCR assays of vacA and cagA genes are listed in Table 1 [14, 15]. DNA samples from H. pylori (D0008, Genekam, Germany) were used as positive control of cagA and vacA genes, and sterile distilled water was used as negative control. PCR was done in 20 µL (for H. pylori) or 25 µL (for vacA and cagA) of total reaction volume containing 1.5 mM MgCl2 (2.0 mM for cagA), 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 200 µM dNTPs each (Fermentas), 0.4 µM primers, 0.3 U of Taq DNA polymerase (Fermentas), and 2 µL (40-260 ng/µL) of DNA. PCR was performed in a DNA Thermal Cycler (Eppendrof Mastercycler 5330, Eppendorf-Nethel-Hinz GmbH, Hamburg, Germany), with 40 cycles for ET2 primer and 35 cycles for vacA and cagA primers. Each cycle consisted of denaturation at 95°C/45 seconds; annealing at 59°C/30 seconds for ET2, 52°C/45 seconds for vacA, and 58°C/45 seconds for cagA; and extension at 72°C/45 seconds. . There was another longer extension of 6 minute at 72°C. PCR products were visualized by electrophoresis in 1% agarose gel, stained with ethidium bromide, and examined under ultraviolet illumination.
The data were then entered and analyzed by using 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 considered statistical significant.
Out of 200 gastric biopsy specimens, 164 (82%) were confirmed to have gastric H. pylori infection by RUT. Seventy nine (48.1%) were male and eighty five (51.8%) were female, with a mean age of 47 years old (range 15 to 88 years old). Sixteen patients (11.8%) had Gastric ulcers, 22 (16.2%) had Duodenal ulcers, 21 (15.5%) had Gastritis, 3 (2.2%) had Gastric cancer, 3 (2.2%) had Duodenit, 34 (25.1%) had Nodularity, 55 patients (40.7%) had None ulcer dyspepsia, 16 (11.8%) had Gastric erythema and 52 patients (38.5%) had Gastric erosion.
Possible combinations of vacA s and m regions were determined in the Iranian population. Overall 63 samples were classified as vacA s1/m1, 73 samples as s1/m2, 22 as s2/m2 and 6 as s2/m1 genotypes. Out of 135 s1 strains, all the samples were successfully sub-types using s1a, s1b and s1c specific primers. Among them 79(48.1%) were s1a positive, 21(12.8%) were s1b positive and 35(21.3%) were s1c positive. In the case of m1 sub-typing the distribution of m1a and m1b was 31.7% and 9.14% respectively. M2 was found in 58.5% of the cases. (Table 2) Data analysis revealed significant association between Duodenal ulcer and s1a/m1a (P=0.04), s1a/m2 (P=0.02) and surprisingly s2m2 (P=0.05) genotypes. There was association between Gastritis disease and s1a/m1a (P=0.01), Nodularity and s1c/m1a (P=0.03), Erythema and s1c/m1a (P=0.05), Gastric erosion and s1a/m1a (P=0.01) genotypes. We didn't find any relationship between Gastric ulcer, Gastric cancer, Duodenit and NUD with vacA genotypes.
Out of 164 ureC- positive samples, cagA gene was detected from 151 (92%). One hundred twenty eight (78.04%) strains with vacA s1 genotype were cagA positive whereas 23(14.02%) strains with vacA s2 genotype were cagA positive, showing that the presence of cagA gene was significantly associated with the vacA s1 genotype (p=0.004). In particular, most samples (95.5%) with the vacA s1/m2 genotype were cagA positive. Also, the prevalence of cagA gene was not related to clinical outcomes ( Table 3).
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The gastric pathogen H.pylori is one of the most genetically diverse bacterial species which may be involved in the complex variety of diseases in infected patients . The geographic distribution of distinct Helicobacter pylori genotypes remains largely unknown and the prevalence of virulent bacterial genotypes in a certain region may have important epidemiological consequences. . The vacA genotypes show considerable variability in different geographic regions. . 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 predominate but s1c is lacking. Our isolates were similar to those from East Asian isolates where s1c is predominant. . Our study showed that Iranian H. pylori isolates are very diverse in genotype and contain the East and the West elements.
H. pylori strains which have cagA gene express and have been considered more virulent than cag- negative strains. . The prevalence of cagA-positive H. pylori varies from one geographic region to another, e.g., 97% in Korea, 94% in Malaysia, 90% in China, 78% in Turkey and 53% in Kuwait. . In this study we found cagA gene in 92% of the H.pylori-positive population. This finding does not substantiate the role of cagA as marked marker for increased virulence, because of high the positivity in all H. pylori strains and this finding is in agreement with studies from East Asia.
A strong association between the cagA and vacA status and peptic ulcer disease has been reported. . Beil et al suggested that the increased inhibitory effect of cagA-positive, cytotoxin-producing strains on mucin synthesis can be considered a possible mechanism responsible for the increased risk of developing peptic ulceration with these H. pylori strains. . Gzyl et al found that cagA gene correlates 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 higher levels of cytotoxin production. . Our data are in agreement with those of Gzyl et aland Beil et al, [7,13] which suggest that H. pylori strains with cagA and vacA s1 genotypes are associated with more severe gastritis.
Investigator's opinions about the association between vacA genotypes and gastric disorders are different. For example in Iran Jafari et al. found no correlation between them, whereas Mohammadi et al and Molaei et al found s1a allele associated with more severe inflammation. [16, 19]. As we described in results, we have found relationship between some diseases and some vacA genotypes but we can'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 associate with NUD diseases, in our evaluation was surprisingly more prevalent in PUD than in NUD patients and had direct relationship with duodenal ulcer. In Thailand, Japan, Korea, Colombia and America no relationship has been found  whereas in Cuba, Lebanon, Hung Kung, England and most of the European countries a significant association between s1 allele and PUD diseases has been reported [22, 23, 24].
We conclude that the dominant genotypes of H.pylori in Iranian patients may be cagA-positive s1/m2. cagA gene positivity rate is probably not closely associated with severity of the disease. H.pylori strains showing vacA s1 genotype are associated with more severe gastritis but this virulence factor can not determine the overall pattern. Therefore, it seems that the nature of the disease complicating chronic infection is determined by host, environmental and bacterial factors and perhaps new virulence factors should be described with more power to discriminate among H.pylori strains