| Background and Aim: H.pylori is associated with various diseases of the upper gastrointestinal tract in children.The main virulence genes of H.pylori include cytotoxin-associated gene(cag A),vacuolating cytotoxin gene A(vac A),and induced by contact with epithelium(ice A).Cytotoxin produced by H.pylori may cause the development of a series of illnesses.With the wide use of the children's H.pylori infection triple therapy eradication,H.pylori resistance rates have continued to increase in recent decades.At present,it is believed that the virulence genotype of H.pylori and the emergence of antibiotic resistant strains influenced the efficacy of eradication therapy.The aim of the study was to undergo H.pylori isolation and culture for gastric mucosa biopsy specimens and antimicrobial susceptibility testing and to examine cag A,vac A and ice A status in the infected children in Jiangxi province,China.The patients with H.pylori infection were treated with eradication.This study was to investigate the incidence of H.pylori and antibiotic primary resistance of strains and analyze the distribution of cag A,vac A and ice A genotypes in H.pylori isolated.We made the study to see whether there was the relationship between it's genotypes and upper gastrointestinal diseases.We explored whether the virulence genotype of H.pylori clinical isolates was related to antibiotic resistance.Furthermore,we observed whether the virulence genotype of H.pylori strains influenced the efficacy of eradication therapy.Materials and methods: 1.Clinical Materials From July 2014 to August 2015,a total of 316 patients with upper gastrointestinal symptoms were included in the study and performed gastroscopy examination and gastric mucosal biopsy.2.H.pylori isolation culture and antimicrobial susceptibility testing After biopsy samples from each patient were crushed and cultured on the surface of plates.The cultured plates were incubated in microaerobic atmosphere conditions.After H.pylori isolation was required,antimicrobial susceptibility testing to amoxicillin,clarithromycin,and metronidazole was performed using the E-test for minimum inhibitory concentration(MIC).Susceptibility testing to furazolidone was carried out using the K-B test.3.PCR amplification of H.pylori virulence genotypes Genomic DNA was extracted from H.pylori strains isolated from gastric biopsy specimens using the QIAamp DNA mini kit(Qiagen Gmb H,Hilden,Germany)according to the manufacturer's instructions.The DNA concentration was determined by spectrophotometry using a Nano Drop 2000(Thermo Scientific,USA).PCR was performed on purified genomic DNA from all H.pylori isolates to examine the presence of virulence genotypes,including cag A,vac A and ice A.Primers were designed based on published papers.The PCR products were electrophoresed on a agarose gel and developed under UV light(Bio-Rad,USA)according to standard procedures.4.Patients with H.pylori infection were performed the triple therapy eradication of Proton pump inhibitor plus two antibiotics.Results: 1.H.pylori isolation culture and antimicrobial susceptibility testing In the present study,316 patients were enrolled in the study.A total of 107 H.pylori isolates(33.9%)were cultured from gastric biopsy tissue,including 81 male and 26 female,ranging from 1 year 9 months to 14 years of age.There were statistically significant change in constituent ratio of the detection rate of H.pylori isolate culture between chronic superficial gastritis groups and duodenal bulb inflammation groups and gastric ulcer groups and duodenal bulb ulcer groups(? 2 = 33.133,P < 0.001).The detection rate of H.pylori isolate culture of patients with duodenal bulb ulcer was higher than patients with chronic superficial gastritis and gastric ulcer(? 2 = 31.861,P < 0.001;? 2 = 1.098,P = 0.295);the detection rate of patients with duodenal bulb inflammation was higher than patients with chronic superficial gastritis and gastric ulcer(? 2 = 8.348,P = 0.004;P = 0.480).In a total of 107 strains of H.pylori isolates,97 strains were tested for antibiotic susceptibilities and the results were read out.Among the 97 H.pylori isolates,the resistance rates of H.pylori to clarithromycin and metronidazole were 20.6% and 47.4%,respectively.The dual-resistance rates to clarithromycin and metronidazole was 12.4%.No H.pylori strains were found to be resistant to amoxicillin or furazolidone.There were statistically significant differences in the resistance rate between the clarithromycin resistance groups and the metronidazole groups(? 2 = 15.524,P < 0.001).2.Association of H.pylori virulence genotypes with gastrointestinal diseases in children Of the total 107 H.pylori isolates,H.pylori cag A genes were detected in 94.4%.In the vac A subtype,the detection rate of vac As1 a and vac Am2 genotypes of H.pylori were the highest,74.8% and 69.2%,respectively,both vac Am1 and vac Am2 were detected in 0.9% of all H.pylori strains;In the chimera of vac A gene,the detection rates of vac As1a/m1,vac As1a/m2 was the highest,51.4%(? 2 = 69.283,P < 0.001);but the vac As1 b and vac As2 subtypes were not detected in any samples.The detection rates of ice A1 and ice A2 genes were 79.4% and 9.3%,respectively(? 2 = 113.822,P < 0.001),and both ice A1 and ice A2 genes were detected in 7.5% of all strains.The detection rates of the genotypes of H.pylori showed no significant differences between the peptic ulcer,chronic gastritis,and duodenal bulbar inflammation groups(P > 0.05).There were no statistical differences in the detection rates of the chimera of cag A/vac As1a/m2 gene between peptic ulcer,chronic gastritis and duodenal bulb inflammation group(? 2 = 2.670,P = 0.263).3.Association of H.pylori virulence genotypes with antibiotic resistance in children There were no statistically significant differences in the detection rate of H.pylori virulence genotypes between the clarithromycin resistant strains,the metronidazole resistant strains,the dual-resistance to clarithromycin and metronidazole and antibiotic sensitive strains(p > 0.05).4.Relationship between of H.pylori virulence genotypes on triple eradication therapy A total of 107 patients were enrolled in this study,9 patients lost to follow-up,98 patients eventually administered with the eradication therapy and reexamined by 13C-urea breath test.By Intention-to-Treat(ITT)and Per-protocol(PP)analyses,H.pylori eradication rate is 64.5% and 70.4%,respectively.There were no statistically significant differences in the detection rate of H.pylori genotypes between the H.pylori successful eradication groups and the unsuccessful eradication groups(p > 0.05).Conclusion: 1.The genotypes of H.pylori are not associated with the type of gastroduodenal disease.No relationship is found between the virulence genotypes of H.pylori clinical isolates and resistance to antibiotics in children.2.The dominant genotypes of H.pylori are cag A,vac As1a/m2,and ice A1,and there are mixed infections with H.pylori strains of different genotypes in infected children in Jiangxi,province,China 3.The resistance rate of H.pylori clinical isolates to clarithromycin and metronidazole is high in Jiangxi province and no resistance to amoxicillin and furazolidone was observed.However,this study detected dual-resistance of H.pylori clinical isolates to clarithromycin and metronidazolei in children.4.H.pylori cag A,vac A and ice A genes are not associated with 14-day omeprazole-based triple therapy on eradication of H.pylori infection in children. |
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