Section of Gastroenterology, Unit Hospitals of Foggia, Foggia, Italy.
Gastroenterology and Digestive Endoscopy, 'Nuovo Regina Margherita' Hospital, Rome, Italy.
J Med Microbiol. 2014 Mar;63(Pt 3):453-457. doi: 10.1099/jmm.0.067942-0. Epub 2013 Dec 16.
Primary clarithromycin resistance is the main factor affecting the efficacy of Helicobacter pylori therapy. This study aimed: (i) to assess the concordance between phenotypic (culture) and genotypic (real-time PCR) tests in resistant strains; (ii) to search, in the case of disagreement between the methods, for point mutations other than those reported as the most frequent in Europe; and (iii) to compare the MICs associated with the single point mutations. In order to perform real-time PCR, we retrieved biopsies from patients in whom H. pylori infection was successful diagnosed by bacterial culture and clarithromycin resistance was assessed using the Etest. Only patients who had never been previously treated, and with H. pylori strains that were either resistant exclusively to clarithromycin or without any resistance, were included. Biopsies from 82 infected patients were analysed, including 42 strains that were clarithromycin resistant and 40 that were clarithromycin susceptible on culture. On genotypic analysis, at least one of the three most frequently reported point mutations (A2142C, A2142G and A2143G) was detected in only 23 cases (54.8%), with a concordance between the two methods of 0.67. Novel point mutations (A2115G, G2141A and A2144T) were detected in a further 14 out of 19 discordant cases, increasing the resistance detection rate of PCR to 88% (P<0.001; odds ratio 6.1, 95% confidence interval 2-18.6) and the concordance to 0.81. No significant differences in MIC values among different point mutations were observed. This study suggests that: (i) the prevalence of the usually reported point mutations may be decreasing, with a concomitant emergence of new mutations; (ii) PCR-based methods should search for at least six point mutations to achieve good accuracy in detecting clarithromycin resistance; and (iii) none of the tested point mutations is associated with significantly higher MIC values than the others.
主要克拉霉素耐药性是影响幽门螺杆菌治疗效果的主要因素。本研究旨在：（i）评估表型（培养）和基因型（实时 PCR）检测在耐药菌株中的一致性；（ii）在方法不一致的情况下，寻找除欧洲报道的最常见突变以外的其他点突变；（iii）比较与单个点突变相关的 MIC 值。为了进行实时 PCR，我们从细菌培养成功诊断为幽门螺杆菌感染且克拉霉素耐药性通过 Etest 评估的患者中检索活检。仅纳入从未接受过治疗且幽门螺杆菌菌株仅对克拉霉素耐药或无任何耐药性的患者。分析了 82 例感染患者的活检，包括 42 株培养时克拉霉素耐药和 40 株克拉霉素敏感的菌株。在基因分析中，仅在 23 例（54.8%）中检测到三个最常报道的点突变（A2142C、A2142G 和 A2143G）中的至少一个，两种方法的一致性为 0.67。在另外 19 个不一致的病例中，又检测到了 14 个新的点突变（A2115G、G2141A 和 A2144T），将 PCR 的耐药检测率提高到 88%（P<0.001；优势比 6.1，95%置信区间 2-18.6），一致性提高到 0.81。不同点突变之间的 MIC 值无显著差异。本研究表明：（i）通常报道的点突变的流行率可能正在下降，同时出现新的突变；（ii）基于 PCR 的方法应至少检测六个点突变以实现克拉霉素耐药性检测的良好准确性；（iii）所测试的点突变均与其他点突变相比没有更高的 MIC 值。
