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目的分析温州地区幽门螺杆菌(Hp)对克拉霉素和左氧氟沙星的耐药情况及相关耐药基因突变特征,同时检测患者的药物代谢酶CYP2C19的代谢类型。通过综合分析药物代谢酶和耐药位点突变情况为临床Hp个体化治疗提供理论依据。方法选择2015年1~3月在瑞安市人民医院经胃镜和病理检查确诊的慢性胃炎患者116例,其中男67例,女49例,年龄24~76岁,平均(43.8±5.3)岁。采用E-test法测定Hp对克拉霉素和左氧氟沙星的最低抑菌浓度(MIC),耐药判定标准:克拉霉素MIC>1μg/m L,左氧氟沙星MIC>1μg/mL。提取Hp基因组DNA,采用PCR法扩增23S r RNA和gyr A基因片段,并对扩增产物进行测序。个体CYP2C19代谢类型的测定同样采用PCR扩增和测序的方法。结果 34株克拉霉素耐药Hp菌株的23S r RNA V区均有基因突变发生,突变位点A2143G最为常见,突变率达到91.17%。左氧氟沙星耐药Hp菌株的gyrA基因最常见的突变方式为N87K(56.76%)。个体药物代谢酶类型主要为快代谢和中代谢类型。结论 23S r RNA基因的突变位点A2143G是导致Hp对克拉霉素耐药的主要原因。gyrA基因的87和91位氨基酸突变是导致Hp对左氧氟沙星耐药的主要原因。临床Hp根除治疗应适时采用高通量检测技术提高个体化用药效能。
Objective To analyze the resistance of Helicobacter pylori (Hp) to clarithromycin and levofloxacin in Wenzhou and the mutation characteristics of related drug resistance genes, and to detect the metabolic type of drug metabolizing enzyme CYP2C19 in patients. Through a comprehensive analysis of drug-metabolizing enzymes and mutations in drug-resistant sites for clinical Hp individualized treatment provides a theoretical basis. Methods A total of 116 patients with chronic gastritis diagnosed by endoscopy and pathological examination in Rui’an People’s Hospital from January to March 2015 were selected, including 67 males and 49 females, aged from 24 to 76 years (average, 43.8 ± 5.3) years. The minimum inhibitory concentrations (MICs) of clarithromycin and levofloxacin against Hp were determined by E-test. The standard of resistance was clarithromycin MIC> 1μg / m L and levofloxacin MIC> 1μg / mL. The Hp genomic DNA was extracted, and the 23S rRNA and gyr A gene fragments were amplified by PCR, and the amplified products were sequenced. Measurement of CYP2C19 Metabolic Type Individual PCR amplification and sequencing are also used. Results The mutations of 23S r RNA V region of 34 clarithromycin - resistant Hp isolates were found. The mutation A2143G was the most common mutation with a mutation rate of 91.17%. The most common mutation of gyrA gene in levofloxacin-resistant Hp strains was N87K (56.76%). The types of individual drug metabolizing enzymes are mainly fast metabolism and moderate metabolic types. Conclusions A2143G, the mutation site of 23S r RNA gene, is the main cause of Hp resistance to clarithromycin. The 87 and 91 amino acid mutations in the gyrA gene are the major causes of Hp resistance to levofloxacin. Clinical Hp eradication therapy should be timely and high-throughput detection techniques to improve individual efficacy.