Department of Molecular Biology, Marilia Medical School, Marilia, São Paulo, Brazil.
BMC Gastroenterol. 2013 Dec 4;13:164. doi: 10.1186/1471-230X-13-164.
Clarithromycin, amoxicillin, and a pump proton inhibitor are the most common drugs recommended as first-line triple therapy for H.pylori treatment, which results in eradication rates close to 80%, varying regionally, principally due to emergency cases and increases of clarithromycin resistant strains. Nucleotide substitutions at the H. pylori domain V of the 23S rRNA fraction are involved in the macrolide resistance and the A2142G and A2143G mutations are predominant in clinical isolates worldwide including in Brazil. As H. pylori culture is fastidious, we investigated the primary occurrence of H. pylori A2142G and A2143G rDNA 23S mutations using a molecular approach directly on gastric biopsies of dyspeptic patients consecutively attended at Hospital das Clinicas of Marilia, São Paulo, Brazil.
Biopsy specimens obtained from 1137 dyspeptic patients, were subjected to histopathology and H. pylori diagnosis by histology and PCR. PCR/RFLP assay was used to detect A2142G and A2143G point mutations at domain V of the H. pylori 23S rDNA associated with clarithromycin resistance. Through the developed assay, a 768 bp PCR amplicon corresponding to1728 to 2495 bp of the 23S H. pylori rDNA is restricted with MboII for A2142G mutation detection and with BsaI for A2143G mutation detection. Occurrence of 23S rDNA A2142G results in two DNA fragments (418 and 350 bp) and of 23S rDNA A2143G results in three DNA fragments (108, 310 and 350pb), due to a conserved BsaI restriction site.
The PCR method used to diagnose H. pylori presented sensitivity, specificity and accuracy of 77,6%, 79,3% and 78,6%, respectively, compared to histology, the gold standard method for H. pylori diagnosis used in our routine. Prevalence of H.pylori with clarithromycin resistant genotypes was 2,46%, with predominance of A2143G 23S rDNA point mutation.
The PCR/RFLP assay was a rapid and accurate H.pylori diagnostic and clarithromycin resistance determination method useful for routine practice. As prevalence of primary resistance of H.pylori to clarithromycin due to A2142G and A2143G mutations remains low in Marilia, the standard clarithromycin containing triple therapy is still valid.
克拉霉素、阿莫西林和质子泵抑制剂三联疗法是治疗幽门螺杆菌的一线治疗方案，根除率接近 80%，但因紧急情况和克拉霉素耐药菌株的增加而在不同地区有所差异。幽门螺杆菌 23S rRNA 结构域 V 上的核苷酸取代与大环内酯类耐药有关，A2142G 和 A2143G 突变是包括巴西在内的全球临床分离株中主要的耐药突变。由于幽门螺杆菌的培养较为复杂，我们采用分子方法直接对巴西马里利亚市临床诊所就诊的消化不良患者的胃活检标本进行检测，以研究幽门螺杆菌 A2142G 和 A2143G rDNA 23S 突变的首次发生情况。
对 1137 例消化不良患者的活检标本进行组织病理学检查和组织学及 PCR 诊断幽门螺杆菌。采用 PCR/RFLP 检测方法检测与克拉霉素耐药相关的幽门螺杆菌 23S rDNA 结构域 V 上的 A2142G 和 A2143G 点突变。通过开发的检测方法，用 MboII 对 A2142G 突变进行检测，用 BsaI 对 A2143G 突变进行检测，对 23S H. 幽门螺杆菌 rDNA 的 1728 至 2495 位核苷酸进行扩增，得到 768bp 的 PCR 扩增子。A2142G 突变导致 23S rDNA 产生两条 DNA 片段（418 和 350bp），A2143G 突变导致 23S rDNA 产生三条 DNA 片段（108、310 和 350bp），这是因为存在一个保守的 BsaI 限制位点。
与组织学（我们常规使用的幽门螺杆菌诊断金标准方法）相比，用于诊断幽门螺杆菌的 PCR 方法的敏感性、特异性和准确性分别为 77.6%、79.3%和 78.6%。具有克拉霉素耐药基因型的幽门螺杆菌的流行率为 2.46%，以 23S rDNA 点突变 A2143G 为主。
PCR/RFLP 检测法是一种快速、准确的幽门螺杆菌诊断和克拉霉素耐药性检测方法，适用于常规检测。由于马里利亚的幽门螺杆菌对克拉霉素的原发性耐药率仍因 A2142G 和 A2143G 突变较低，因此标准的克拉霉素三联疗法仍然有效。
