Peng Xianhui, Song Zhiqiang, He Lihua, Lin Sanren, Gong Yanan, Sun Lu, Zhao Fei, Gu Yixin, You Yuanhai, Zhou Liya, Zhang Jianzhong
State Key Laboratory of Infectious Disease Prevention and Control, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Chinese Center for Disease Control and Prevention, Beijing, China.
Department of Gastroenterology, Peking University Third Hospital, Beijing, China.
Int J Med Sci. 2017 May 15;14(6):595-601. doi: 10.7150/ijms.18996. eCollection 2017.
A gastric juice-based real-time polymerase chain reaction (PCR) assay was established to identify infection, clarithromycin susceptibility and human genotypes and to guide the choice of proton pump inhibitor (PPI), clarithromycin and amoxicillin treatment for tailored eradication therapy. From January 2013 to November 2014, 178 consecutive dyspeptic patients were enrolled for collection of gastric biopsy samples and gastric juice by endoscopy at the Peking University Third Hospital; 105 and 73 -positive and -negative patients, respectively, were included in this study. infection was defined as samples with both a strongly positive rapid urease test (RUT) and positive histology. A series of primers and probes were distributed into four reactions for identifying the gene coupled with an internal control ( gene), A2142G and A2143G mutants of the gene, and single-nucleotide polymorphisms (SNPs) G681A of and G636A of . The E-test and DNA sequencing were used to evaluate the clarithromycin susceptibility phenotype and genotype. The SNPs and were also evaluated by nucleotide sequencing. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of this gastric juice-based real-time PCR assay were evaluated by comparing with the same measures obtained through gastric biopsy-based PCR and culture. The diagnostic sensitivities of the culture, PCR, and gastric biopsy- and gastric juice-based real-time PCR assays were 90.48% (95/105), 92.38% (97/105), 97.14% (102/105) and 100% (105/105), respectively; the specificities of the above methods were all 100%. Higher false-negative rates were found among the gastric biopsy samples assessed by culture (10.48%, 11/105), PCR (7.62%, 8/105) and real-time PCR (2.86%, 3/105) than in gastric juice by real-time PCR. Regarding clarithromycin susceptibility, a concordance of 82.98% (78/94) and discordance of 17.02% (16/94) were observed among the different methods, discrepancies that mainly represent differences between the clarithromycin susceptibility phenotype and genotype. Three coinfections of susceptible and resistant strains were detected, with resistant-to-susceptible ratios of 1.16, 3.44, and 8.26. The genotyping results from gastric juice by real-time PCR were completely in accordance with those obtained from biopsy samples by conventional PCR. This gastric juice-based real-time PCR assay is a more accurate method for detecting infection, clarithromycin susceptibility and polymorphisms. The method may be employed to inform the choice of proton pump inhibitor (PPI), clarithromycin and amoxicillin treatment for tailored eradication therapy.
建立了一种基于胃液的实时聚合酶链反应（PCR）检测方法，用于鉴定感染、克拉霉素敏感性和人类基因型，并指导选择质子泵抑制剂（PPI）、克拉霉素和阿莫西林进行个性化根除治疗。2013年1月至2014年11月，北京大学第三医院连续纳入178例消化不良患者，通过内镜检查收集胃活检样本和胃液；本研究分别纳入了105例阳性和73例阴性患者。幽门螺杆菌感染定义为快速尿素酶试验（RUT）强阳性且组织学检查阳性的样本。一系列引物和探针被分配到四个反应中，用于鉴定幽门螺杆菌基因并结合内部对照（管家基因）、幽门螺杆菌基因的A2142G和A2143G突变体，以及空泡毒素基因的单核苷酸多态性（SNP）G681A和细胞毒素相关基因A的G636A。采用E试验和DNA测序评估幽门螺杆菌克拉霉素敏感性表型和基因型。空泡毒素基因和细胞毒素相关基因A的SNP也通过核苷酸测序进行评估。通过与基于胃活检的PCR和培养获得的相同指标进行比较，评估了这种基于胃液的实时PCR检测方法的敏感性、特异性、阳性预测值（PPV）和阴性预测值（NPV）。培养、PCR以及基于胃活检和胃液的实时PCR检测方法的幽门螺杆菌诊断敏感性分别为90.48%（95/105）、92.38%（97/105）、97.14%（102/105）和100%（105/105）；上述方法的特异性均为100%。与基于胃液的实时PCR相比，通过培养（10.48%，11/105）、PCR（7.62%，8/105）和实时PCR（2.86%，3/105）评估的胃活检样本中发现的假阴性率更高。关于克拉霉素敏感性，不同方法之间的一致性为82.98%（78/94），不一致性为17.02%（16/94），这些差异主要代表了幽门螺杆菌克拉霉素敏感性表型和基因型之间的差异。检测到3例敏感菌株和耐药菌株的混合感染，耐药与敏感比例分别为1.16、3.44和8.26。基于胃液的实时PCR基因分型结果与通过常规PCR从活检样本中获得的结果完全一致。这种基于胃液的实时PCR检测方法是检测幽门螺杆菌感染、克拉霉素敏感性和多态性的更准确方法。该方法可用于指导选择质子泵抑制剂（PPI）、克拉霉素和阿莫西林进行个性化根除治疗。
