CYP2C19 Genotype, CagA Genotype and Antibiotic Resistant Strain of Helicobacter pylori Infection

Background: H. pylori is a class I carcinogen and major cause of gastric cancer. Few previous studies reported relationship between H. pylori infection, CYP2C19 genotype and functional dyspepsia (FD) subtype. The aim of this study was to determine relationship between CYP2C19 genotype and FD subtype patients(host factor) with antibiotic resistant strains of H. pylori infection and CagA genotype(bacterial factor). Methods: FD patients who were investigated with gastroscopy at Thammasat University Hospital, Thailand during March 2017-November 2017 were enrolled. Two antral gastric biopsies were obtained for rapid urease test, E-test and cultures. CagA genotypes (CagA1a and CagA2a) were determined by PCR and CYP2C19 genotype was determined by PCR-RFLP. FD patients were categorized as epigastric pain syndrome(EPS) and postprandial distress syndrome (PDS). Results: 93 FD patients with H. pylori infection were enrolled (37 male, 56 female, mean age 54.5 years). There were 33 patients with EPS and 60 patients with PDS. CYP2C19 genotype revealed 55.9% rapid metabolizer (RM), 40.9% intermediate metabolizer (IM) and 3.2% poor metabolizer (PM) genotypes. Antibiotics susceptibility tests demonstrated 62.8% resistant to metronidazole, 12.9% resistant to clarithromycin and 27.1% resistant to fluoroquinolone. CagA 1a and CagA 2a were demonstrated in 6 patients(11.5%) and 46 patients(88.5%). CagA2a genotype was more prevalent in PDS than EPS patients (94.3%vs.76.5%; P =0.08) without significance. In intermediate metabolizer (IM), CagA2a genotype was significant higher in PDS than EPS(100% vs.25%; P=0.004). Conclusions: PDS, CYP2C19 RM genotype and CagA 2a gene of H. pylori infection were the predominant type of FD in Thailand. Metronidazole remain the most common antibiotic resistant strain of H. pylori infection in FD patients. PDS (host factor) was significantly related to CagA2a genotype (bacterial factors) only in patients with intermediate metabolizer. Appropriate dose of proton pump inhibitor (PPI) and correct regimens for H. pylori eradication in FD patients should be consider to improve clinical outcomes.


Introduction
H. pylori causes chronic gastric inflammation and lead to peptic ulcer disease (PUD), mucosa associated lymphoid tissue (MALT) lymphoma and gastric cancer (Marshall and Warren, 1984;Li et al., 2014;Miftahussurur et al., 2017Vilaichone et al., 2018). The International Agency for Research in Cancer classified H. pylori as a type I carcinogen and previous studies has found that H. pylori treatment could be reduced the incidence of gastric cancer (Vilaichone et al., divided into CagA 1a and CagA 2a. CagA 1a strain of H. pylori demonstrated more virulence and associated with more gastric inflammation due to activation of proinflammatory cytokines eg. IL-1B, IL-6 and IL-8 (Yamaoka, 2010;Vilaichone et al., 2011) Pharmacogenetic polymorphisms are long known affecting biotransformation and clinical outcome. The clinical importance of these variants depends on allele-frequency and the effect size of the clinical outcome parameters. The proton pump inhibitor (PPI) drugs are affected by genetic polymorphisms of the cytochrome P450 drug metabolizing enzyme CYP2C19. The outcome of CYP2C19 genotype testing were demonstrated as: rapid metabolizer (RM), intermediate metabolizer (IM) or poor metabolizer (PM). CYP2C19 genotypes have effect on pharmacokinetics and pharmacodynamics of PPI which involved in changing gastric pH and effectiveness of H. pylori eradication (Phiphatpatthamaamphan et al., 2016) Currently, H. pylori resistance rates to antibiotics are becoming problem in most parts of the world. H. pylori antibiotic resistance caused eradication rates have been declining while the prevalence of antibiotic resistance rates have been increasing (Malfertheiner et al., 2017) In Thailand, Antibiotic resistance was presented in 50.3% including amoxicillin 5.2%, tetracycline 1.7%, clarithromycin 3.7%, metronidazole 36%, ciprofloxacin 7.7%, Levofloxacin 7.2% and multi-drugs in 4.2% (Vilaichone et al., 2013). The aim of this study was to determine relationship between CYP2C19 genotype and FD subtype of patients (host factor) with antibiotic resistant strains of H. pylori infection and CagA genotype (bacterial factor).

Patients
Eligible patients' age between 18-70 years have been evaluated by gastroscopic examination at Thammasat University Hospital, Pathumthani, Thailand between March 2017 and November 2017. After endoscopy, those with diagnosis of functional dyspepsia (FD), which was established during gastroscopy with normal finding or mild gastritis, were considered entering in this study. FD patients were diagnosed by Rome IV diagnostic criteria (Stanghellini et al., 2016) and categorized as epigastric pain syndrome (EPS) and postprandial distress syndrome (PDS). Exclusion criteria included patients with (1) a history of previous H. pylori eradication, (2) on anticoagulants drugs (3) previous gastric surgery, (4) administration of antibiotics, bismuth and PPI drugs in the prior 4 weeks, (5) pregnancy and breast-feeding woman, (6) allergy to one of the study medication, and (7) other serious comorbidities (such as major systemic diseases, cardiovascular disease, malignancies). All patients provided written informed consent.
During the endoscopy, 2 biopsy samples from gastric antrum and body were obtained for rapid urease test, H. pylori culture and histological examination. H. pylori culture were obtained by inoculating the specimens on nonselective media (Columbia agar supplemented with 7% sheep blood) under a microaerophilic atmosphere for at least 7 days. The colonies were identified by gram straining, oxidase, catalase and urease production. Susceptibility for metronidazole was tested by Epsilometer test (E-test) or GenoType®HelicoDR.

H. pylori CagA genotyping
Chromosomal DNA was selected from plate cultures expanded from each single H. pylori colony using the QIAamp Tissue kit (QIAGEN Inc. Santa Clarita, CA, USA) according to the manufacturer's instructions. Polymerase chain reaction (PCR) amplification was done for 35 cycles consisting of 1 min at 95°C, 1 min at 52°C and 1 min at 72°C. Final cycle included a 7-min extension step to confirm full extension of PCR products. CagA genotypes (CagA1a and CagA2a) were also determined by technique of PCR.

CYP2C19 genotype
DNA was extracted from gastric tissue by using QIAamp Tissue kit (QIAGEN Inc. Santa Clarita, CA, USA) and forward to perform PCR-RFLP with gel electrophoresis in order to identify difference in the CYP2C19 genotypes. The results of CYP2C19 genotype testing were expressed as: rapid metabolizer (RM), intermediate metabolizer (IM) or poor metabolizer (PM).

Statistical analysis
The association between FD, CagA genes, antibiotic resistance and CYP2C19 genotype were evaluated by using chi-squared. Association between categorical variables were determined using odds ratio and 95% confidence interval (95% CI). The P values < 0.05 were considered to be statistically significant. All analyses were performed by using SPSS Statistics version 23.0 (IBM Corp., Armonk, NY). The study was conducted according to good clinical practice guidelines and was approved by our university ethics committee.  Table 1.

Total
CagA2a genotype was more common in PDS than EPS patients (94.3% vs. 76.5%; P = 0.08), but without statistical significance. Both FD subtypes were not related antibiotics susceptibility tests and CYP2C19 genotype as demonstrated in Table 2. CYP2C19 genotype was not related antibiotics susceptibility tests and CagA genotype as demonstrated in Table 3. In each CYP2C19 genotype, antibiotic susceptibility test of H. pylori was not significantly associated with FD subtypes of patient. In subgroup analysis of patients with intermediate metabolizer (IM), CagA2a genotype was significantly higher in PDS than EPS (100% vs. 25%; P = 0.004) as demonstrated in Table 4.

Discussion
The majority of FD patients in Asian countries were PDS subgroup (Kawamura et al., 2001 Table 4. Subgroup Analysis of CagA Genotype and FD Patient in Each CYP2C19 Genotype also demonstrated that PDS was predominant in our population. In Thailand, metronidazole resistance was found to be as high as in 11-36%, whereas fluroquinolone and clarithromycin resistance were 7-29% and 4-5 %, respectively (Sahara et al., 2012;Chotivitayatarakorn et al., 2017). In our study, antibiotics susceptibility tests demonstrated 62.8% resistant to metronidazole, 12.9% resistant to Clarithromycin and 27.1% resistant to fluoroquinolone. CagA is a highly immunogenic protein encoded by the CagA gene. CagA plays major roles in gastric inflammation and injury in association with activated inflammatory cells infiltration. H. pylori strains isolated from Western and East Asian countries could be easily distinguished by PCR-based CagA 5' and 3' region genotyping, and named the East Asian type H. pylori strains as CagA 1a type and the Western type H. pylori strains as CagA 2a type. East-Asian type CagA is considered to be more toxic than its Western homologues and more strongly associated with severe clinical outcomes, including gastric cancer. (Sahara et al., 2012;Uchida et al., 2015) Recent study, prevalence of CagA 1a genotype was demonstrated in 11.6-24.6% in Thailand. (Vilaichone et al., 2011;Uchida et al., 2015) In our study, prevalence of CagA 1a genotype was demonstrated in 11.5%. Prevalence of CagA 1a genotype was lower than other Asian country. Also, the incidence of gastric cancer in Thailand was lower than other Asian populations.
Functional dyspepsia (FD) is common disease. The Rome IV Consensus proposed the subdivision of FD into two subgroups: the epigastric pain syndrome (EPS) and the postprandial distress syndrome (PDS). The pathophysiology is complex and multifactorial. The relationship between H. pylori infection and FD remains unclear, but there is evidence showing benefit of eradicating H. pylori in patients with chronic dyspepsia, with number needed to treat of 14. Cost-effectiveness analyses suggested that H. pylori eradication is the most cost-effective treatment for infected dyspeptic patients compared with alternative therapies that need to be taken long-term (Stanghellini et al., 2016). The term H. pylori-associated dyspepsia is recently introduced and defined as dyspepsia with long-term sustained remission of symptom after successful eradication of H. pylori (Sugano et al., 2015;Suzuki and Mori, 2015). Previous study showed that the prevalence of Cag A positive strains was not significant in PDS patients in Thailand (Vilaichone et al., 2011). Recent study from Taiwan reported association between H. pylori infection and FD patients with only pure PDS, but not with pure EPS or overlap syndrome. There was more prevalence of Cag A positive strains in PDS patients (Fang et al., 2015). In this study, we found equal prevalence of CagA positive H. pylori infection in PDS and EPS patients, since all patients in our study are CagA positive. CagA 2a were more prevalent in PDS compared with EPS patients without statistical significance, but CagA 2a were significantly more common in PDS in subgroup analysis of patients with IM. However, it should be noted that the magnitude of association between Cag A 2a positive strains and PDS symptom from subgroup analysis, even with statistical significant, could be resulted from unmeasured confounders. A further study is warranted to confirm the relationship between CagA genotype and FD in Asian populations.
In conclusions, PDS, CYP2C19 RM genotype and CagA 2a gene of H. pylori infection were the predominant type of FD in Thailand. Metronidazole resistance remain the most common antibiotic resistant strain of H. pylori infection in FD patients followed by fluoroquinolone resistant strain. PDS (host factor) was significantly related to CagA2a genotype (bacterial factors) only in patients with intermediate metabolizer. However, there were no association between FD subtype and CYP2C19 genotype or antibiotic resistance pattern of H. pylori infection in Thailand. Host and bacterial factors play important roles in pathogenesis of FD patients and might help to management for better outcomes in this particular disease. Appropriate dose of proton pump inhibitor (PPI) and correct regimens for H. pylori eradication in FD patients should be consider to improve clinical outcomes.