Helicobacter pylori vacA genotyping in relation to cagA status , ultrastructure of gastric mucosa and clinical outcomes in Egyptian patients

Helicobactor pylori (H. pylori) has been strongly associated with gastritis, peptic ulcer and is linked to an increased risk of gastric cancer. The cytotoxin-associated gene product (cagA) and the vacuolating cytotoxin (vacA) have been implicated as two major virulence factors of H. pylori. Since there is an increasing evidence that genetic variability of H. pylori may have clinical importance, we aimed to evaluate different vacA genotypes and reveal its relationship with endoscopic and transmission electron microscopy (TEM) findings among H. pylori infected Egyptian patients. Forty H. pylori infected patients possessing vacA gene who underwent upper endoscopic examination were considered to be infected with H. pylori when rapid urease test and detection of 16S rRNA in gastric biopsy recorded positive. Both vacA and cagA genotypes were detected by polymerase chain reaction (PCR). The TEM was performed to assess the ultra-structure of the gastric mucosa. Four vacA genotypes were identified, the most prominent was the s2/m2 allele combination (52.5%) followed by s1/m1 (27.5%), s1/m2 (17.5%) and s2/m1 genotype was found just in one H. pylori strain (2.5%). There were significant correlations between vacA s2/m2 and gastritis (65.2%), and vacA s1/m1 and peptic ulceration (57%). The cagA gene was associated with 38% of vacA genotypes and 60% of which were significantly associated with vacA s1/m1 genotype with the development of severe gastritis reaching up to gastric ulcer. The TEM revealed H. pylori spiral and coccoid forms, cytoplasmic vacuolar degeneration caused by vacA, swollen mitochondria and dilated rough endoplasmic reticulum. In Egypt where prevalence of H. pylori infection is high, genotyping of H. pylori virulence factors can help to predict patients who are at a high risk of related gastroduodenal diseases. Although H. pylori with vacA s2/m2 genotype is mostly related to low level of virulent strains yet, significant crosstalk between H. pylori strains harboring both vacA s1/m1 and cagA gene provides crucial insights into virulence of high level.


INTRODUCTION
Helicobacter pylori is a spiral, Gram-negative bacterium that inhabits the stomachs of approximately half of the world's population (Warren and Marshall, 1983).Infections with H. pylori may induce gastritis, gastric and duodenal ulcers and even is linked to an increased risk of gastric cancer (Khalifa et al., 2010).H. pylori secrete many of the proteinaceous factors that are important for initial colonization and subsequent persistence in the stomach.Two major virulence factors of H. pylori have been described; the cytotoxin-associated gene product (cagA) and the vacuolating cytotoxin (vacA).Both play a crucial role in determining the clinical outcome of H. pylori infection and their genes could serve as epidemiological markers (Marie, 2012).
The VacA toxin binds to target cells and is internalized causing severe "vacuolation" that has been attributed to the formation of VacA anion selective channels in membranes.In addition to the induction of vacuolation, VacA exerts a variety of other effects on target cells, including disruption of mitochondrial functions, stimulation of apoptosis and blockade of T-cell proliferation (Palframan et al., 2012).
The vacA gene, encoding the vacuolating toxins is virtually present in all strains of H. pylori.Polymorphism in vacA gene sequence has been identified in three variable regions; signal (s) region, mid (m) region and intermediate (i) region.Two types of allelic variations in the s-region and m-region are classified as s1 or s2 and m1 or m2 respectively.Therefore, the vacA gene of a given strain is composed of any of the possible combination of s and m region sequence type (Atherton et al., 1995).Type s1/m1 vacA causes more epithelial cell damage than type s1/m2, whereas type s2/m2 and the rare s2/m1 are non-toxic (Letley et al., 2003;Argent et al., 2008).In our study we aimed to evaluate different vacA genotypes and to reveal its relationship with endoscopic and TEM findings among H. pylori infected Egyptian patients.

Patients and specimens
Forty H. pylori infected patients possessing vacA genes who underwent upper endoscopy for various dyspeptic symptoms at Endoscopy Unit, Theodor Bilharz Research Institute (TBRI) Hospital in-between March 2012 to April 2013 were enrolled in this study.The mean age of the patients was 50.05 years (range, 17-76 years), 29 were males and 11 were female.None of the patients had received non-steroidal anti-inflammatory drugs, as well as antibiotics, H2 receptors antagonists or proton pump inhibitors in the past four weeks prior to the study.Thorough endoscopic examination of the oesophagus, stomach and duodenum and clinical condition of the patient (gastritis, peptic ulceration, normal endoscopy and other findings) were assessed.Four antral biopsy specimens were obtained from each patient.This study was approved by TBRI Institutional Review Board (IRP) FWA 00010609 and informed consent was obtained from each subject before endoscopic examination.A patient was considered to be infected with H. pylori when rapid urease test and detection of 16S rRNA in gastric biopsy specimen were recorded positive.

Rapid urease test for H. pylori infection
One gastric biopsy was inserted and completely immersed in the reagent solution which is composed of urea, phenol red and stabilizers (Bussero, Milan, Italy).The positive sample turns color from yellow to magenta within 30 min.

DNA extraction
Two antral gastric biopsy specimens for each patient were stored in 0.9% saline at -70°C until used for polymerase chain reaction (PCR) assay.DNA for PCR was extracted directly from antral gastric biopsy specimens using the QIAamp tissue kit provided by (Wizard SV Genomic DNA Purification System, USA) according to the manufacturer's instruction.The DNA extracts were stored at -20°C until used for PCR assays.

PCR-based H. pylori detection, vacA genotyping and cagA status
PCR assays were performed in a volume of 50 μl with approximately 10 μg of total extracted DNA.PCR amplifications were carried out in Gene Amp PCR system 9700 (Bio Rad T100 thermal cycler).The 50μl reaction mixture consisted of 10 ug of the extracted DNA, x1 PCR buffer, 1.5 mM Magnesium Chloride, 200 μM of each dNTP, 20 pmol of each primer (Table 1) and 1U Taq DNA polymerase (Promega).

PCR amplification of the H. pylori 16S rRNA
Amplification was carried using the following cycling parameters: An initial denaturation at 95°C for 5 min and 35 cycles of 95°C for 30 s, 60°C for 30 s and 72°C for 30 s.This was followed by a final extension of 72°C for 10 min (Chisholm et al., 2001).

PCR amplification of the H. pylori vacA genotypes
The following cycling parameters were used: an initial denaturation at 95°C for 4 min and 35 cycles of 95°C for 1 min, 52°C for 1 min and 72°C for 1 min.This was followed by a final extension of 72°C for 10 min (Falsafi et al., 2009).

PCR amplification of the H. pylori cagA gene
The following cycling parameters were applied: an initial denaturation at 94°C for 5 min and 35 cycles of 94°C for 1 min, 59°C for 1 min and 72°C for 1 min.This was followed by a final *Corresponding author.E-mail: manal_kandil@hotmail.com.Fax: 35427901.extension of 72°C for 10 min (Falsafi et al., 2009).Each PCR product was separated on a 2% Agarose gel and 50 bp ladder was used as DNA molecular weight standard.In each PCR assay, negative control (lacking DNA) was included.

Transmission electron microscopy (TEM) examination of gastric mucosa
Segments from each gastric biopsy specimens were collected from H. pylori infected patients.Segments were immediately fixed for 2 h in equal volumes of glutaraldehyde 4% and caccodylate 0.2 M. The fixed segments were washed in equal volumes of Sacchrose 0.4 M and caccodylate 0.2 M for 2 h and incubated in osmium tetroxide 2% and caccodylate 0.3 M for 1 h.The samples were washed with distilled water and finally dehydrated in ascending grades of ethyl alcohol for 5 min each (30, 50, 70 and 90%) then absolute alcohol 100% for 10 min for three times.Substitution in a mixture of epoxy resin and ethyl alcohol in equal volumes for 1 h was done.Impregnation in pure resins using Epon A and Epon B in equal volumes making three washes on three successive days was done.The specimens were embedded in epoxy resin to which was added an accelerator DMP 30 in special capsules then left in oven at 60°C for 2 days to polymerize and harden.The thin sections were stained with uranyl acetate and lead citrate and were examined with TEM to assess the interaction between H. pylori and the ultra-sructure of the gastroduodenal epithelial cells (Bai et al., 2010).

Statistical analysis
Results are expressed as number (%).Comparison between categorical data was performed using Chi square test.Statistical Package for Social Sciences (SPSS) computer program (version 19 windows) was used for data analysis.P value ≤ 0.05 was considered significant and < 0.01 was considered highly significant.

RESULTS
H. pylori infection in all patients was confirmed by rapid urease test and detection of 16S rRNA of H. pylori by agarose gel electrophoresis (110 bp) in gastric biopsy specimens (Figure 1).

Clinical outcomes
During upper gastrointestinal endoscopy of the studied 40 H. pylori infected patients possessing vacA genes, their gastroduodenal mucosa had developed gastritis in 23 (57.5%), whereas 7 (25%) had peptic ulceration (including one with suspected malignant ulcer).Other endoscopic findings as; oesophageal varices, gastrooesophageal reflux, gastric prolapse, hiatus hernia were revealed in 9(22.5%)cases and only one patient had normal gastric mucosa.

Relationship of vacA genotypes to cagA status and clinical outcomes
The cagA gene was detected in 38% (15/40) of the studied vacA genotypes strains (Figure 3); 60% of which were significantly associated with vacA s1/m1 genotype (P=0.041) with the development of severe gastritis reaching up to gastric ulcer, 33.3% (5/15) of cagApositive strains were associated with vacA s1/m2 genotype and only one strain was of s2/m2 genotype.

Analysis of TEM examination
Electron microscopy revealed H. pylori in its spiral and coccoid forms (Figure 4), as well as cytoplasmic vacuolar degeneration played by the vacuolating toxin (Figure 5).Ultrastructural examination of the tiny gastric biopsies revealed tissue debris of exfoliating degenerated mucosal epithelium with exposure of blood vessels of the lamina propria in many examined ultrathin sections with large lipid accumulation.Inflammatory lymphocytic cells was evident in the examined sections.Curved H. pylori and cluster of unidentified bacilli nearby degenerated mucosal gastric structures were depicted in H. pylori positive cases.Also, gastric mucous cells with their characteristic granules resting on intact lamina propria were the main cells seen in most of the examined sections.Many of them showed vesiculated endoplasmic reticulum and vacuolated cytoplasm.

DISCUSSION
Little is known about the geographic distribution of H. pylori genotypes in distinct regions, particularly in Egypt.
The vacA genotypes and its relationship to clinical outcomes, cagA status and TEM findings were investigated in H. pylori strains from 40 infected Egyptian patients possessing vacA gene.Different results have been reported in studies related to vacA s and H. pylori strains.In the current study, the vacA s2 allele was the most predominant genotype (55%) followed by vacA s1 (45%).Our findings are similar to an earlier report from Egypt (Van Doon et al., 1999), Korea (Kim et al., 2001) and Turkey (Ozbey and Aygun, 2012).Al Quabandi et al. (2005) reported that North Africans   were predominantly infected with the s2 type.In contrary to our results, the previous study from Kuwait reported that vacA s1 and s2 types were detected in approximately equal numbers in biopsies obtained from patients of Middle Eastern origin.Previous reports from Cyprus (Krashias et al., 2013) and Saudi Arabia (Sugimoto et al., 2009) revealed that s2 is the main allele among their H. pylori strains.The m-region encodes the vacA binding site to host cells, which appears to be more effective in the m1 than in m2 forms (Ferreira et al., 2014).Similar to previous studies in Turkey, Iraq, Iran, Saudi Arabia (Hussein, 2010) and China (Chung et al., 2010), m2 was predominantly found in all studied H. pylori strains (70%) previously reported.However, this was inconsistent with previous results obtained from Egypt where equal distribution of vacA m1 and m2 was found (Amer et al., 2013).Genetic variation within virulence factors may account for differences in the pathogenic properties of strains, and thus may help to explain the discrepancies between the number of infected individuals and those that end up developing gastric cancer (Ferreira et al., 2014).
The allele combination between vacA s and vacA m genotypes and their association to gastroduodenal disorders differ greatly (Nimri et al., 2006).In this study, the prevalence of the vacA genotypes s1m1 was 27.5%, and 57.1% of which was highly significant in relation to peptic ulcer.This is in agreement with previous studies in which association between this genotype and severe gastric outcomes was recognized (Marie, 2012).It is worth to mention that, in these cases long curved virulent spiral forms of H. pylori were detected at the level of electron microscopy in association with ulcerated and degenerated mucosal lining.Also, multiple bacterial microorganisms exhibiting curved appearance were seen among the mononuclear inflammatory cells undergoing apoptotic changes and in between the debris of degenerated mucosal cells.It was reported that the long spiral form facilitate the penetration and movement of the microorganism through the mucous gel.H. pylori lacking the spiral form loses its infectiousness (Bai et al., 2010).Moreover, in this study cytoplasmic vacuolar degeneration of mucosal cells with swollen mitochondria and dilated rough endoplasmic reticulum denoting the presence of the vacA gene toxic effect was an important finding.(Atherton et al., 1995) and Bai et al. (2010) reported that the pivot role in cell damage induced by H. pylori is played by vacuolating toxin.
The significant association between vacA s1/m1 genotypes and gastric carcinoma development has been substantiated by meta-analysis using reports of patients from diverse geographic origin, including Western, Middle East, Latin America and Africa countries.In an observational longitudinal study from Spain, the progression of premalignant lesions was more frequent in patients infected with vacA s1/m1 strains than those infected with less virulent vacA s2/m2 strains (Ferreira et al., 2014).Our data emphasizes the significant association of the most predominant (52.5%) allele combination s2/m2 with gastritis (65.2%).This predominance goes in accordance with Benenson et al. (2002) and El-Gharbawy et al. (2006).This similarity in H. pylori genotypes in three neighboring countries, Egypt, Jordan, and Gaza strip, indicates a geographic influence, which was reported by Abu Amra (2010).Whereas in Egypt, Amer et al. (2013), reported that all possible combination of vacA s1 with m were recognized in their work, in addition, H. pylori virulence could not be predicted in relation to different genotypes.
Regarding the relationship between H. pylori genotypes and clinical outcome, we found a significant association between cagA status and vacA s1/m1 genotype with development of severe gastritis reaching up to gastric ulcer.These findings are in agreement with Marie (2012) and Ozbey and Aygun (2012).
The attribution between the presence of cagA and severe clinical outcome has been a controversial point; as it was previously found that colonization with cagA positive strains has been associated with a fivefold increased risk for diagnosis of duodenal or gastric cancer (Diab et al., 2009).Whereas in other studies the occurrence of gastric malignancy was independent of cagA status and others implicated the pivotal roles of other virulence factors (cag E, cag T, vac A and bab A) in the aetiology of gastric cancer (Marie, 2012).

Conclusion
In Egypt where prevalence of H. pylori infection is high, genotyping of H. pylori virulence factors can help to predict patients who are at a high risk of related gastroduodenal diseases.Although H. pylori with vacA s2/m2 genotype is mostly related to low level of virulent strains yet, significant crosstalk between H. pylori strains harboring both vacA s1/m1 and cagA gene provides crucial insights into virulence of high level.

Figure 5 .
Figure 5. Electron micrograph of H. pylori infected gastric mucosa showing cytoplasmic vacuolar degeneration (arrow) caused by the vacuolating toxin.

Table 1 .
Primer sequences and size of expected amplicon of each PCR assay.

Table 2 .
Prevalence of vacA genotype alleles among the studied 40 H. pylori strains.

Table 3 .
Relationship between H. pylori vacA genotypes and clinical outcomes in 40 H. pylori infected patients.