Prevalence of H. pylori strains harbouring cagA and iceA virulence genes in saudi patients with gastritis and peptic ulcer disease

Aim: The study is aimed to detect the presence of cagA, iceA1, and iceA2 virulence genes in H. pylori from gastric biopsies, and to deduce the correlation between these genotypes and the two clinical outcomes peptic ulcer disease (PUD), and gastritis. Materials and methods: Thirty three Saudi patients 15 males and 18 females, 20 to 90 years were assigned into two groups PUD and gastritis. Genomic DNAs were extracted from biopsy specimens and used to detect the presence of cagA, and iceA genes by PCR typing system. Fisher’s and Phi coefficient association tests were used for statistical analysis. Results: Genotyping show that both cagA and iceA genes were amplified from 27 specimens (81.7%). The prevalence of cagA+ and cagAgenotypes or iceA+ and iceAgenotypes did not differ significantly between males and females (p=0.070). Within the PUD and gastritis groups, the percentages of specimens positive for cagA gene were 76.9 % and 85 %, while those positive for iceA+ were 92.3% and 75% respectively. All cagA+/iceA+ combined genotypes was statically correlated with peptic ulcer (77%). This correlation was not observed within H. pylori specimens typed from gastritis. Patients with either PUD or gastritis were most likely infected by several strains of H. pylori. Conclusion: Different strains of H. pylori have virulent genotypes evidenced by PCR-based genotyping from biopsy specimens at a reasonable cost and time. These virulence strains spread at Taif province, may result in sever clinical outcomes such as ulcers which may be developed to cancer, the situation which necessitates further studies.


Introduction
Helicobacter pylori colonizing the human stomach acquired by contaminated water or food or poorly disinfected endoscopes. Lifetime persistence of this organism within the host could result in a number of gastroduodenal diseases ranging from mild gastritis, atrophic gastritis, and peptic ulcer disease to malignant diseases such as gastric adenocarcinoma and Mucosa-Associated-Lymphoid-Tissue (MALT) Lymphoma [1,2].
Although a chronic active gastritis will be developed by most of infected patients, the majorities of infections are asymptomatic [1][2][3]. Found that, 15-20% of infected patients will develop gastric or duodenal ulcer disease and less than 1% will develop gastric adenocarcinoma. Due to poor correlation between symptoms and disease, many of gastric cancer cases are detected lately when the disease is rooted and become incurable. Direct PCR methods performed on H. pylori DNA isolated from biopsy specimens have been evaluated previously [4][5][6][7].
In developing countries, H. pylori infection is particularly high (up to 80%) [9][10][11]. The prevalence of H. pylori infection in Jordan and Bahrain was 77.5% and 79%, respectively [12,13]. In Kuwait and Egypt, H. pylori were present in 84% and 86% of the biopsy samples, respectively [14,15] while the rate of infections reached 87% in the Eastern region and 61.6% in Central and Western region in the Kingdom of Saudi Arabia [16,17]. Although some studies have reported an excess of H. pylori in one gender versus the other [18,19], found no gender differences in H. pylori prevalence overall.
Over the past few years, research on pathogenicity markers has become gradually more important and intense in an attempt to detect bacterial strains associated with each of these diseases. The cytotoxin-associated gene A (cagA gene) was the first virulence factor detected in H. pylori strains. This gene encodes a protein that is associated with an increase in intensity of gastric inflammation and, consequently, with severe clinical outcomes, inducing an intense inflammatory process, with dense neutrophil infiltration, which can cause serious hurt to the gastric mucosa. The induced by contact with epithelium (iceA) gene has two allelic forms, iceA1 and iceA2 [18,19].
The expression of iceA1 was controlled by contact between H. pylori and human epithelial cells [20] and the iceA1 genotype was associated with enhanced mucosal interleukin (IL)-8 expression and acute antral inflammation [21].
Although iceA gene has no correlation with gastric cancer  [20,22], proved the role of iceA1 allele in peptic ulcer, others did not find any role for this allele in gastroduodenal disease [22] while, [23] reported an inverse association between the iceA2 allele and peptic ulcer. Several studies were not able to explain the role of iceA and its correlation with clinical outcomes in other populations; therefore the mechanism of how iceA induce PUD remains unclear [24,25]. Such contradicted results between the iceA genotype and clinical consequences could be explicated by the genetic diversity or differences in the geographic region, which were previously reported for other virulence factors [26]. Moreover, geographic variations in addition to genetic heterogeneity of the host further contribute to the diversity of host responses to particular H. pylori strains and genotypes [27].
The objectives of current study are to detect the virulence genes (cagA, iceA1 and iceA2) by polymerase chain reaction (PCR) in gastric biopsy specimens and to find out the possible association between these virulence genotypes and the clinical outcomes.

Sample collection
Thirty three biopsy specimens were collected from 15 males and 18 females attending the endoscopy clinic at three hospitals; King Faisal, AL Hada Armed Forces, and King AbdulAziz in Taif province, Kingdome of Saudi Arabia between September 2011 and February 2012. Mean age (± 47) was varying from 20 to 90 years. These patients were scheduled for gastroscopy by their physician based on various symptoms such as abdominal pain, reflux and dyspepsia.
The study was approved by the ethics committee of Taif University and each hospital has obtained an informed consent from each patient prior to performing the study.
The gastric biopsies were transferred immediately into sterile tubes containing 3 ml of saline or Brucella broth labeled with the patient's I.D. and date. Samples were brought directly to the lab for immediate processing. Campylobacter-like organism(CLO) test was done on the 33 mucosal specimens. Patients were assigned in the following groups based on the gastroenterologist's diagnosis: 1. PUD group: when there was evidence of erosion or ulceration in the gastric mucosa with exudates and erythema. 2. Gastritis group: when there was evidence of inflammation, edema, punctuate hemorrhage, friability, or nodularity.
The biopsy specimens were fragmented using a sterile pestle and mortar. Further homogenization was done by passing the lysate alternatively 5 times through a 0.9 mm needle (20 gauges) fitted to a syringe. The resulting lysate was divided into aliquots and placed into a microcentrifuge tube for DNA extraction.

Genomic DNA isolation
Genomic DNAs were extracted from thirty three biopsy specimens using the QIAamp DNA mini kit (Qiagen GmbH, Hilden, Germany), as described by the manufacturer. The tissue biopsies were centrifuged at 5000 xg for 10 min and re-suspended in 200 µl of ATL buffer (supplied in the QIAamp DNA Mini Kit) for complete lysis. Finally, the DNAs were eluted in100 µL of elution buffer. DNA purity and quantity was determined using a GeneSys 10UV spectrophotometer (Thermo Scientific, USA).

Genotyping-PCR
Isolated genomic DNAs (gDNAs) were used to detect the presence of cagA, and iceA by PCR. Primers used for cagA gene amplification were as follows: forward primer cagA F1

Statistical analysis
Fisher's exact and Phi coefficient association tests were used for analysis of two-by-two and two-by-four-tables of categorical data. All tests were two-tailed, and the significance level was assumed as 0.05.

Results
The distribution of cagA+, and cagA-genotypes within collected samples is shown in (Table 1 and Figure 1) (15/20) for PUD and gastritis cases, respectively. The percentage of iceA+ genotype within each clinical outcome was significantly higher than that of iceA-genotype. The percentages of iceA+ genotype differed significantly between the two clinical outcomes, as iceA+ genotype was detected more frequently in PUD patients as compared with gastritis patients. As with cagA gene, there was no association between iceA genotypes and gender of patients.
In the current study, 93.9% (31/33 cases) of H. pylori positive specimens had at least one of the two virulence genes examined in this study, i.e., cagA and/or iceA (Figure 1). This suggests that the majority of H. pylori isolates examined in this study had virulence potential as evidenced by PCR-based molecular testing.
The prevalence of the combined cagA and iceA genotypes among the 13 peptic ulcer and 20 gastritis cases is shown in Figure 1. The percentage of cagA+/iceA+ genotype was significantly high (p<0.001) within peptic ulcer (76.9%) and also within gastritis (65%) cases. However, no association was revealed between the prevalence of the four genotypes (++, +-, -+, --) and the clinical outcome by using 2x4 Fisher's exact test (p=0.498). Figure 1 shows that all of the cagA+ H. pylori specimens (n=10) that were typed from peptic ulcer cases were also found to have the iceA+ genotype. Of the 17 cagA+ H. pylori specimens that were typed from gastritis patients, 13 specimens had the iceA+ genotype. The cagA+ genotype, therefore, could be a predictive marker for the iceA+ genotype in H. pylori specimens isolated from peptic ulcer patients. This association was not observed within H. pylori specimens typed from gastritis cases.
The occurrence of iceA1 and iceA2 double positive genotypes within the studied samples are shown in Table 2. Out of the 33 samples examined 19 (58%) were double positive for iceA1 and iceA2 genes. These iceA1 and iceA2 double positives were found in 11 PUD (11/13=85%) and 8 gastritis (8/20=40%) cases. Table 2 shows also that cagA/iceA1/iceA2 positives occurred in 77% (10/13 cases) of PUD cases and in only 40% (8/20 cases) of gastritis cases. There was a significant association between the occurrence iceA1 and iceA2 double positives and PUD (p=0.0188). Thus, it appears that infection with multiple strains of H. pylori occurs more frequently in patients with PUD, compared to those with gastritis.

Discussion
Our study determined the cagA and iceA geneotype of H. pylori biopsy samples in a group of patients attending the endoscopy clinic at Al-Hada Armed Forces Hospital, King Faisal Hospital, and King Abdul Aziz Hospital.
In this study, PCR was used to characterize H. pylori infections diagnosis gender iceA2 iceA1 cagA Samples No.

Gastritis cases (n = 20)
:  in biopsy specimens and to examine the association between genotypes and clinical outcomes. The cytotoxin associated gene A (cagA gene) has been proposed as a marker for a genomic pathogenicity island (cag-PAI) of approximately 40 kbp whose presence is associated with more severe clinical outcomes [28,29]. The induced by contact with epithelium gene (iceA gene) has recently been discovered [20]. The two main allelic variants of the gene are iceA1 and iceA2. The expression of iceA1 is upregulated on contact between H. pylori and human epithelial cells, and may be associated with peptic ulcer disease [24,[30][31]. The results of the current study indicated that 93.9 % of H. pylori isolates examined had at least one of these two virulence genes as evidenced by PCR-based molecular testing. These results were in agreement with that obtained by [32]. Our data indicated that the incidence of H. pylori-related diseases has been observed to be similar among men and women and no statistically significant difference in prevalence based on gender. However [33,34], reported that the rate of infection with H. pylori, afflict men more frequently than women studied among 556 African-Americans. In a study reported by [35] the prevalence rate among males (18.9%) was significantly higher (p<0.001) than that among females (9.0%).
The cagA gene was detected in 81.8% (27/33) of recovered H. pylori specimens which is similar to other countries [36,37].
The frequency of cagA gene was reported to be around 62% in a Saudi study [38] compared to 70% in Europe, 85% in Estonian and Russia, 90% in East Asia [39] and 63% in Japan [40]. The percentage of cagA+ genotype within each clinical outcome was significantly higher than that ofcagA-genotype (p<0.001). However, the prevalence of cagA+ and cagA-genotypes did not differ significantly between the two clinical outcomes (p=0.658).
Likewise, the iceA gene was detected in 81.7 % (27/33), while 6 cases (18.8 %) were iceA-by PCR (Figure 1). The percentage of iceA+ genotypes were 92.3% (12/13) and 75% (15/20) for PUD and gastritis cases, respectively. The percentage of iceA+ genotype within each clinical outcome was significantly higher than that of iceA-genotype. The percentages of iceA+ genotype differed significantly between the two clinical outcomes, as iceA+ genotype was detected more frequently in PUD patients as compared with gastritis patients. As with cagA gene, there was no association between iceA genotypes and gender of patients.
In a study reported by [41] found that 87.4% of the positive H. pylori cases were iceA2 positive compared to only 12.6% cases positive for iceA1. As reported by [42,43] iceA1 expression is associated with a higher activity of the gastric inflammation, a condition that increases the risk for developing ulcer disease and gastric carcinoma.
Previous studies in the United States andthe Netherlands have demonstrated a strong association between iceA1 and ulcer disease which also proved by [30,31,41].
The prevalence of the combined cagA and iceA genotypes among the 13 peptic ulcer and 20 gastritis cases is shown in Figure 1. The percentage of cagA+/iceA+ genotype was significantly high (p<0.001) within peptic ulcer (76.9%) and also within gastritis (65%) cases. However, no association was revealed between the prevalence of the four genotypes (+ +, + -, -+, --) and the clinical outcome by using 2x4 Fisher's exact test (p=0.498). Figure 1 shows that all of the cagA+ H. pylori specimens (n=10) that were typed from peptic ulcer cases were also found to have the iceA+ genotype. Of the 17 cagA+ H. pylori specimens that were typed from gastritis patients, 13 specimens had the iceA+ genotype. The cagA+ genotype, therefore, could be a predictive marker for the iceA+ genotype in H. pylori specimens isolated from peptic ulcer patients. This association was not observed within H. pylori specimens typed from gastritis cases.
The occurrence iceA1 and iceA2 double positive genotypes within the studied samples are shown in Table 2. Out of the 33 samples examined 19 (58%) were double positive for iceA1 and iceA2 genes. These double positives were found in 10 PUD (11/13=77%) and 8 gastritis (8/20=40%) cases. Thus, it appears that infection with multiple strains of H. pylori occurs more frequently in patients with PUD, compared to those with gastritis. Out of the 19 iceA1 and iceA2 double positive samples, 18 samples had the cagA+ genotype. Table 2 shows that cagA+/iceA1/iceA2 double positive genotypes occurred doi: 10.7243/2052-6180-2-2 in 77% of PUD cases and in only 40% of gastritis cases.
These results are in agreement with those reported by [33] who found a high correlation between the iceA1+ and peptic ulcer disease. Also as reported by [39], all the ulcer cases (100 %) were iceA1 positive with statistically significant correlation (p=0.0001), while iceA1 allele was found in 94.6% of gastritis cases. Other studies from Asia suggested no association between cagA & iceA genotypes and peptic ulcer disease [44]. As investigated by [45] H. pylori genotypes are not equally distributed all over the world.
In conclusion, PCR-based genotyping should be done for high-risk patients who are infected with multi genotypes of H. pylori in order to prevent the development of ulcer and cancer diseases later in their life.