抗生素耐药基因位点和药物代谢酶联合检测在幽门螺杆菌根除治疗中的应用

Application of combined detection of antibiotic resistance gene loci and CYP2C19 in helicobacter pylori eradication therapy

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〉Itzg／mL，左氧氟沙星MIC〉1μg／mL。提取Hp基因组DNA。采用PCR法扩增23SrRNA和gyrA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23SrRNAV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23SrRNA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 investigate drug resistance of Helicobacter pylori （Hp） strains to levofloxacin and clarithromycin in Wenzhou district, and to detect the type of CYP metabolism, so as to provide theoretical basis for clinical anti-Hp treatment. Methods 116 patients with chronic gastritis were selected in Rui＇an People＇s Hospital from January 2015 to March 2015 and their diagnosis under gastroscope and pathology were analyzed. The patients included 67 cases of male, 50 cases of female, aged from 24 to 76 years old, average （43.8±5.3） years old. Minimum inhibitory concentration （MIC） values for elarithromycin and levofloxaein resistance in Hp were tested by E-test method. Genomic DNA of Hp was extracted, followed by PCR amplification and DNA sequencing for 23S rRNA and gyrA fragments. Determination of CYP2CI9 metabolism type was also performed by the methods of PCR amplification and sequencing. Results 34 Clarithromycin resistant Hp strains showed mutations in the 23S rRNA V region, most commonly seen at Site A2143G （91.17%）. Gene gyrA of the levofloxacin-resistant strains was mostly mutated as N87K （56.76%）. The types of individual drug metabolizing enzymes were mainly fast metabolism and medium metabolism. Conclusion A2143 mutation of 23S rRNA leads to the Hp resistance to clarithromycin. The 87 and 91 amino acid mutations of gyrA gene are the main reasons for the Hp strains resistance to levofloxacin. The eradication therapy of Hp infection should be based on the high throughput detection technology to improve the effectiveness of individual drug use.