Introduction

Helicobacter pylori is a gram-negative bacterium that persistently colonize the human gastric mucosa (^{1, 2, 3,  4}). Gastric colonization by H. pylori account as a risk factor for the development of peptic ulcer disease and distal gastric adenocarcinoma ^{(5, 6 ,7}). The genetic diversity is consistently detected among H. pylori strains isolated from infectious patients and this genetic diversity can helps to account for varying clinical outcomes among persons colonized with H. pylori (^{8, 9}). Candidate markers for distinguishing disease-associated H. pylori strains (i.e., those associated with peptic ulceration or gastric adenocarcinoma) from less virulent strains include presence of the cag pathogenicity island and s1/m1 polymorphisms in vacA alleles (¹⁰). Most of studies survey relation of several H. pylori virulence factors simultaneous with clinical diseases (^{11, 12, 13, 14}). In this way, survey of prevalence hops, vacA toxin and secretion.

system type IV (in this case cagA) genes can lead to important findings in associated with clinical outcomes of H. pylori infection ^{(15, 16, 17 and 18}).  The outer membrane proteins as H. pylori virulence factors have a high level of genetic diversity (^{19, 20}). Hops (H. pylori outer membrane proteins) such as Bab, Sab, Alp, HopZ and HopQ have difference prevalence in H. pylori strains isolated from difference geographical area and clinical outcomes

(^{21,22,23,24,25,26,27and 28}).hopQ I allele is predominantly associated with presence of cagA in Eastern Asian strains isolated from patients with peptic ulcer and hopQ II allele is predominantly associated with absence of cagA in Western strains isolated from patients (^{29, 30 and 31}). It is noticeable, how is genetical pattern of hopQ alleles in H. pylori strains isolated from Iranian patients as a country between western and eastern countries.  There is high rates of H. pylori infection in Iranian population and increase the number of gastrointestinal complaints led to study whether the presence of the Hop alleles can result in clinical outcomes (^{29, 30 and 31}). As a considerable point, it is shown HopQ as an outer membrane protein lead to decline attachment of H. pylori to gastric epithelial cells that effect on delivery of CagA protein into gastric epithelial cells presumably ^{(32, 33, 34 and 35}). In previous studies it shown presence of hopQ alleles is associated with cagA situation (36, 37, 38, 39, 40 and 41). So, determine prevalence of hopQ alleles and cagA together in Iranian strains can support or decline previous studies. The main objective of this study was to evaluate the association between the presence of cagA, vacA and hopQ alleles in H. pylori strains isolated from patients with peptic ulcer using the PCR method.

Materials and Methods

Persons with sadness in the area of ​​the upper gastrointestinal and referred to Tohid Hospital at Sanandaj were objective cases. 100 Gastric biopsy samples were collected from patients with Peptic ulcer and H. pylori positive using Endoscopy. Situation of infection to H. pylori determined with use urea breath test. Information on factors such as age, sex and drugs obtained by questionnaire and were prepared for all forms of morality with peptic ulcer patients. Pathological diagnosis or procedure was performed by a specialist. At least two Gastric biopsies were collected from each patient for test RUT (Rapid urea test) and molecular and pathological study. To investigate the molecular sample taken after the transition to 1.5 microliter tubes containing thioglycollate medium and transfer to Microbiology Lab. in Kurdistan University of Medical Sciences and were stored in a freezer at -80°C for DNA extraction time. For DNA extraction stage, biopsy samples were transferred in sterile conditions and carefully. Then QIAamp DNA Mini Kit (Qiagen) as DNA extraction kit were used for DNA extraction directly from biopsy samples. DNA extraction was performed such as company protocols in catalog. Specific primers were used for hopQ I/II, vacA and cagA as shown in Table 1. The final volume of PCR master mix was 25 ml and was prepared using Master kit (Cinnagen, Iran). Thermal cycling for PCR amplification was optimized as shown in Table 2. PCR products rounded on 2% agarose gel and stained with Sybeer green (Cinnagen, Iran), then observed by UV (Ultraviolet) light. Chi-square test (version 19, SPSS Inc., Chicago, IL) was used for analyses statically.

Table 1: Primer Designer

Table 2: Program thermocycler instruments

Results

The study of 100 Gastric biopsy samples from Iranian patients with Peptic ulcer and H. pylori-positive shown frequency of genes hopQ I, hopQ II, cagA, vacA s1, vacA s2, were 54 (54%), 46 (46%), 51 (51%), 83 (83%), 17 (17 percent) respectively (Table 3). Results of Chi-square test statistically shown a significant correlation between the presence of the hopQ I and cagA genes (P<0.01), hopQ I and vacA s1 genes (P<0.026) and vacA s2 and hopQ II in cagA-negative strains (Table 4).

Table 3: Frequency of hopQ, vacA and cagA Genotypes in Peptic ulcer patient

*(n=100)

Table 4: Relationship between hopQ Alleles and cagA and vacA

*𝑃 value of <0.05 was considered as statistically significant

Discussion

Progress by the interaction of several factors in the pathogenesis of H. pylori infection as well as infection by Helicobacter pylori host of superficial inflammation of the stomach and inevitable role in the etiology of peptic ulcer disease show (^{10, 15, 23 and 38}). Access to the successful colonization of the biological concepts for a bacterial adhesion mechanism is complex and bacterial produced the largest share among all products should be made to account for bacterial colonization (^{1, 2, 3 and 4}). The hopQ gene in H. pylori is a major protein of the outer membrane (^{29, 33}). To determine the relationship between HopQ protien and gastrointestinal diseases may be to provide a point to answer questions about the adhesion of the bacteria to the stomach epithelial cells (^{30, 31 and 36}). The study to determine the frequency of genotypes associated hopQ (hopQ alleles with cagA and hopQ alleles with vacA) in H. pylory strains isolated from biopsy samples was designed. It is believed that the analysis based on the genotyping of H. pylori strains isolated from clinical specimens can be useful in prognosis of Infectious Disorders (^{36, 37 and 41}). According to the results of recent studies hopQ I allele correlated significantly with an increased risk of developing Stomach ulcers in Western countries and also hopQ II allele are checked in this population frequently (³⁰). Sicinschi and colleagues in 2012 assessed the frequency of allele’s hopQ, vacA and cagA in H. pylori strains isolated from 86 asymptomatic children. The frequency of allele’s hopQ I and vacA was detected 73.7% and 91.7% respectively (P<0.0001) (³⁶). In 2013, Belogolova and colleagues presented a study on protein secretion system that HopQ protein as a virulence factor associated with CagA protein and the type 4 secretion system of H. pylori and answers related to effect of CagA protein on cell morphology and cell dispersion. According to the results, HopQ protein as a cofactor plays an important role in the type 4 secretion system of H. pylori (34). In our study, the frequency of allele’s hopQ I, vacA and cagA were detected in H. pylori strains isolated from 100 peptic ulcers samples. The relationship between the presence of hopQ I and cagA (p<0.01) and also hopQ I and vacA s1 (p<0.026) was significant statistically. It can because to presence of hopQ gene on genetic area of cagA and vacA virulence genes and simultaneous expression of these genes is developing a stomach ulcer argue. Further studies in this direction would be to identify strategies for physicians and the anti-peptic ulcer disease, gastritis and gastric cancer to improve.

Acknowledgements

The authors wish to thank H. Ghafori, Sh Fakhari, A. ahmadi, R Ramazanzadeh, MB Khademerfan, B Nikkhou, F fathi, A jalili for their kind assistance in this study. This research has been supported by Kurdistan University of Medical Sciences Services grant

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