Prevalence of H . pylori in gastric biopsy specimen in the southeastern region of Turkey

Introduction: Helicobacter pylori is a Gram-negative, microaerophilic bacterium that colonizes human gastric mucosa. Gastric ulcer, duodenal ulcer, chronic atrophic gastritis, mucosa-associated lymphoid tissue lymphoma, and stomach adenocarcinoma are associated with H. pylori as the etiological agent. Cytotoxin-associated gene A (cagA), which is one of the most important virulence factors of H. pylori, encodes a 120– 145 kDa protein. The prevalence of cagA genes shows differences in H. pylori infections based on geographical area, and cagA-positive H. pylori strains play an important role in pathogenesis of gastric carcinoma. Methodology: The aim of this study was to detect the prevalence of cagA and vacA genes in H. pylori isolates in adult patient groups in the southeastern region of Turkey. The presence of H. pylori was investigated in gastric biopsy specimens using the culture method, and polymerase chain reaction (PCR) analysis was performed to detect the presence of the cagA and vacA s1 genes. Results: H. pylori was detected in 65% (84/129) of patients who had gastrointestinal complaints. The number of vacA s1 and cagA genes of isolates were 44 (74.5%) and 31 (52.5%), respectively. Conclusions: H. pylori infection in southeastern region of Turkey with are comparable to those in developed countries. Patients with cagAand vacA-positive H. pylori have a higher risk of severe inflammation and atrophy and should therefore be monitored for the development of gastric cancer.


Introduction
In many parts of the world, most gastric ulcer, duodenal ulcer, and chronic gastritis patients are infected with H. pylori.The organism plays a major etiological role in the development of gastric mucosaassociated lymphoid tissue (MALT) lymphoma, and distal gastric cancer [1,2].It has been reported that 50% of adults in developed countries and 80%-90% of the population in developing countries are infected with H. pylori [3,4].Along with the antigen test on stools, urea breath test and endoscopic and histopathological examination are considered the gold standards for diagnosis [5].
There are different virulence factors affecting changes that may occur in the gastric mucosa of patients with H. pylori.Vacuolating cytotoxin A (vacA), cytotoxin-associated gene A (cagA) protein, and urease enzyme are the most important virulence factors of H. pylori.Recent studies have focused on investigating the relationship between virulence factors and clinical condition [6][7][8].CagA protein is present 60%-80% in H. pylori strains and is generally accompanied by VacA [9].
The H. pylori status of the population of western and central Turkey is largely known, but the data from southeastern Turkey is not clear, so this study was planned and conducted to investigate the prevalence of H. pylori in urban and rural populations in the southeastern region of Turkey.The relationship between H. pylori genotypes and clinical outcome was evaluated by investigating the presence of cagA and vacA s1 genes in H. pylori strains isolated from patients with chronic gastritis in Diyarbakır Research and Training Hospital.

Patients and sampling
A total of 129 gastric dyspeptic patients (64 male, 65 female) who were admitted to

H. pylori cultivation and identification
After biopsies were taken, samples were collected in Brucella broth medium (Becton Dickinson, New Jersey, USA) with 20% glycerol.All homogenized gastric biopsy specimens were inoculated onto H. pylori-selective agar medium (bioMérieux, Marcy I'Etoile, France).Inoculated plates were incubated at 37°C in an atmosphere of 5% O2, 10% CO2, and 85% N2 for 5-7 days.Bacterial colonies were identified as H. pylori on the basis of colonial morphology, positive urease, catalase and oxidase tests, and Gram stain.All isolated H. pylori strains were stored at -80°C in Brucella broth medium with 20% glycerol.

Genomic DNA extraction
At the time of DNA extraction, genomic DNA was obtained in all isolates using a bacterial genomic DNA extraction kit (NanoHelix, Daejeon, South Korea).A 399 bp fragment of the 16S rRNA gene from the culture was amplified by polymerase chain reaction (PCR) using the Helicobacter genus-specific primers HeliF (AAC GAT GAA GCT TCT AGC TTG CTA G) and HeliR (GTG CTT ATT CST NAG ATA CCG TCA T) [10].The presence of H. pylori was confirmed by culture and PCR.

Analysis of cagA and vacA s1 status in H. pylori
PCR analysis was performed on H. pylori DNA samples to detect the presence of the cagA and vacA s1 genes.For detection of the cagA gene, primers CAGAF and CAGAR, which yield a fragment of 349 bp from the middle conservative region of the cagA gene, were used.For analysis of the vacA s1 region, primers VA1-F and VA1-R were used.Primers VA1-F and VA1-R yielded a fragment of 259 bp for s1 variants.The following protocol was used to amplify the genes: initial denaturation for 2 minutes at 95°C for 1 cycles; 20 seconds at 95°C for 30 cycles, 40 seconds at 52°C for 40 cycles, and 1 minute at 72°C; and a final elongation for 5 minutes at 72°C.After PCR, the amplified PCR products were electrophoresed in 1% agarose gels and examined under UV illumination (Figure 1).Specific primers were used to detect cagA and vacA s1 genes in all isolates (Table 1).

Results
A total of 84 (65%) H. pylori strains were isolated by culture examination of biopsy specimens from 129 patients (64 male, 65 female).These patients had complaints about acute or chronic gastritis.Genotyping was performed on 59 H. pylori strains isolated from 33 male and 26 female patients.The vacA s1 gene was detected in 44/59 (74.5%) and cagA in 31/59 (52.5%) of the isolates.Both genes were found to be positive in 25/59 patients.

Discussion
Mucosal damage in stomach mucosa occurs in patients with H. pylori.VacA encodes a vacuolating toxin that is released by H. pylori and injures epithelial cells.VacA protein stimulates vacuolization in epithelial cells under in vitro conditions.These mechanisms have importance in the pathogenesis of stomach cancer.
With activation in low pH, VacA protein plays a role in connecting to epithelial cells.Recent studies have provided new insights into how VacA action at mitochondria might be functionally associated with cell death.VacA was demonstrated to disrupt mitochondrial dynamics [11][12][13].Additionally, there was a significant correlation between vacA s1 type and enhanced chronic inflammation.The vacA s1 positivity rate was 74.5% in all patients.
When CagA protein secreted by H. pylori is injected into a host cell, toxic change is stimulated.CagA can disrupt signaling pathways by phosphorylationdependent and -independent mechanisms, leading to abnormal proliferation, motility, and cytoskeletal change in gastric epithelial cells [14][15][16].
Strains that contain the cagA gene may lead to more severe clinic cases by stimulating cell transformation [16].According to recent studies, there is an EPIYA-C containing region in H. pylori isolates that have cagA genes [17].The EPIYA-C motif has a dual function in membrane association and tyrosine phosphorylation.It has been suggested that the EPIYA-C motif is a crucial therapeutic target of cagA-positive H. pylori infection [18].
Karrlson et al. studied H. pylori genotype in 155 gastric biopsy specimens.Their findings showed that two or more cagA EPIYA-C motifs are related to atrophy in gastric mucosa [19].
The course of H. pylori infection is almost certainly determined by a combination of host, bacterial and environmental factors, such as immune response, bacteria's virulence factors and the stomach acidic enviroment [20].According to World Health Organization (WHO) statistics, risk for cancer development is doubled in persons infected with H. pylori.Additionally, H. pylori is reported as a class 1 carcinogen in gastric adenocarcinoma [21].Risk for gastric cancer development is 2% among H. pyloriinfected people [22].
The prevalence of cagA-positive H. pylori strains may vary depending on geography and patients' ages.Data from a study conducted by Gunn et al. [23] indicated that 91/120 (76%) isolates were positive for H. pylori strains by culture in England in 1998.Many other studies have also found that cagA is mostly accompanied by vacA genotype s1.Our findings are consistent with these results.Researchers in many studies have shown that the same factors (cagA and s1, i1 and m1 vacA) are associated with gastric cancer and precancerous intestinal metaplasia [24][25][26][27][28].
Also, Plummer et al. [29] researched the existence of the cagA gene in precancerous gastric lesions and found that the prevalence of cagA was 86% in the group with more severe lesions and 59% in the group with less severe lesions, based on 2,145 biopsy specimens in Venezuela.
Preneoplastic lesions were tracked by periodically conducting biopsies on 312 patients in Spain between 1988 and 2007.When results were evaluated, H. pylori strains harboring cagA, vacA s1, and vacA m1 genotypes were found more frequently in patients with more advanced gastric preneoplastic lesions [30].
H. pylori strains positive for the cagA gene (242/286; 85%) were detected in Alaskan patients between 1998 and 2005 [31].Of these patients, 220 were native and 66 were non-native.The infection ratio of the isolates that had the cagA gene was 198/220 (90%) in natives and 44/66 (67%) in non-natives (p < 0.0001), and indicates geographical and ethnic differences.Researchers have reported that these results are close to those obtained from Europe and North America.
In an Italian population, vacA s1, i1, and m1 strains had all been reported to be significantly associated with gastric cancer (GC) [33].Rhead et al. [32] reported similar findings in Iran.Basso et al. [33]   Italy.According to their results, cagA-positive strains were significantly associated with GC and PU.GC risk was further associated with the number of cagA EPIYA-C segments.An increasing number of EPIYA-C segments also increased the risk of intestinal metaplasia.In this study's findings, type s1 and i1 vacA alleles were also associated with GC and type i1 vacA with PUs and duodenal ulcers.
The "Asian paradox" that has been described by researchers might be explained by the widespread prevalence of weakly cytotoxic strains and correspondingly low frequency of H. pylori-associated diseases.However, here, the prevalence of strains with the vacA s1 region among Southeast Asia populations was very high in East Asian and Latin American populations, who have a higher risk of gastric carcinogenesis [34][35][36].
In the South Korean population (n = 225), the majority of H. pylori strains carry the vacA s1/i1/m1 allele and the cagA EPIYA-ABD allele.These facts may contribute to the high incidence of gastric maladies, including gastric cancer.Indeed, the finding that the majority of South Korean H. pylori strains carry the most toxic forms of cagA and vacA may explain the reason for the high prevalence of gastric disease and mortality among patients with gastric cancer in South Korea [37].
A total of studies with a combined 1,281 patients (4 reports from Vietnam, 4 from Thailand, 5 from Malaysia, and 1 from Singapore) were included in the systematic analyses [38].Of the 13 included studies, 93% of H. pylori strains were cagA positive.This genotype positivity rate was higher than the one found in our study.Both Western-and East Asian-type strains of H. pylori are found in Southeast Asia and are predominantly cagA-positive and vacA s1 type.However, the frequency of H. pylori infection in Turkey is lower than in the Asia region.In Southeast Asia, patients infected with vacA m1 type or cagA-positive strains have an increased risk of peptic ulcer disease.This vacA s1 genotype was highly prevalent in our patients who had peptic ulcers.The presence of cagA may be useful in prognosis.
In a study that included 450 patients with gastric symptoms, H. pylori was detected in biopsy specimens of 201 (45%) patients via PCR [39].While 32% of patients had normal gastric mucosa, 68% had different gastroduodenal lesions.In this study, the prevalence of cagA gene was 52% (104/201), in which 97 were Western types and 3 were East Asian.The s1m1 genotype, which is a dominant vacA type, was predominantly detected in H. pylori-positive cases.
Researchers associated higher and lower rates in previous studies conducted in different regions of Pakistan with geography.
In H. pylori strains recently isolated from biopsies of 46 patients, 37 (80.4%) of the 46 H. pylori clinical isolates were positive for cagA [40].Additionally, the cagA gene was mostly accompanied by vacA s1m1 genotype in these isolates.
In Kuwait, cagA, vacA s1, and vacA s2 genes were found in 52.5%, 44.4%, and 39.4% of the patients, respectively, while 10.1% were positive for both vacA s1 and vacA s2 [41].The cagA gene was found in combination with vacA s1 (cagA + vacA s1) in 31.3% of the patients.Patients with the vacA s1 gene alone also had a significantly more moderate-marked degree of chronic inflammation, while those with vacA s2 had significantly less chronic inflammation.
The presence of the vacA s1 genotype of H. pylori is associated with more severe chronic inflammation and atrophic gastritis.Our findings are consistent with this knowledge.
The prevalence of H. pylori infection was 58.9% in the Dominican Republic [42].The H. pylori infection rate in patients with peptic ulcers (82.6%) was significantly higher than in patients with gastritis (54.5%).In this study, cagA-positive/vacA s1m1 genotype was the most prevalent.Patients with cagApositive H. pylori could be at higher risk of severe inflammation and atrophy.

Conclusions
This study analyzed the relationship between gastritis and vacA s1 and cagA positivity and demonstrates the prevalence of cagA and vacA s1 in clinical isolates of H. pylori in the southeastern region of Turkey.

Figure 1 .
Figure 1.The s region was identified by amplification with primers VA1-F and VA1-R.The vacA s1 region produced a 259-bp amplicon and cagA region produced a 349-bp amplicon.
Gastric biopsy specimens were collected from the antrum and corpus of the stomach.No subjects had received treatment for H. pylori infection.The local ethics committee approved the protocol of genotype research.

Table 1 .
Polymerase chain reaction (PCR) primers for amplification of cagA and vacA sequences.
a Nucleotide positions in the cagA gene of H. pylori ATCC 53726 (GenBank accession no.L117714); b Nucleotide positions in the vacA gene of H. pylori 60190 (GenBank accession no.U05676).
studied the vacA signal, mid and intermediate region polymorphisms, cagA presence, and EPIYA-C segment number by PCR in 203 H. pylori-infected subjects (53 gastric cancer, 52 peptic ulcer [PU], and 98 gastritis) in

Table 2 .
Association of cagA and vacA s1 gene positivity with gastric disease.