克拉霉素耐药幽门螺杆菌株的基因型分布

背景：根除幽门螺杆菌（H.pylori）治疗在临床上的应用日益普遍。耐药菌株的出现是近年H.pylori根除率下降的主要原因,尤其是目前根除治疗作用最强的抗生素之一——克拉霉素。目的：研究克拉霉素耐药H.pylori菌株的基因型分布,为快速检出抗生素耐药提供基础。方法：以琼脂稀释法筛选出2002年9月～2003年2月13株原发性、22株获得性克拉霉素耐药H.pylori菌株,提取基因组DNA。聚合酶链反应（PCR）-反向斑点杂交法检测克拉霉素耐药H.pylori菌株23SrRNA基因中7种不同的点突变（A2115G、G2141A、A2142G、A2142C、A2143G、A2143C和A2142T）。结果：34株（97.1%）克拉霉素耐药H.pylori菌株发生A2143G突变,其中13株为原发性,21株为获得性;1株（2.9%）获得性耐药菌株发生A2142G突变。结论：我国克拉霉素耐药H.pylori菌株基因型以23SrRNA基因A2143G突变占主导地位,与欧美国家报道的A2142G和A2143G突变率相近不同。     

粪便基因型检测幽门螺杆菌对克拉霉素耐药性的研究

背景：幽门螺杆菌（Hp）耐药情况日趋严重,选择快速、敏感、价廉的分子生物学技术对Hp耐药进行检测具有重要的临床意义。目的：评价检测粪便Hp基因突变对诊断克拉霉素耐药的有效性,并探讨cagA基因与耐药的相关性。方法：纳入74例13C-尿素呼气试验阳性患者,采集其新鲜粪便标本,提取粪便DNA,采用巢式PCR法扩增Hp23SrRNA,采用PCR—RFLP法检测限制性内切酶BbsI、BceAI、BsaI对23SrRNA扩增产物的酶切情况,采用PCR法扩增cagA基因。结果：74例患者的粪便标本中,60例扩增出Hp23SrRNA367bp片段,其中17例可被BsaI酶切,60例均未被BbsI、BceAI酶切。cagA阳性、阴性表达者的23SrRNA突变率相比差异无统计学意义（P〉0．05）。结论：通过粪便基因型检测Hp对克拉霉素耐药是快速、简便的方法。江苏地区Hp对克拉霉素的耐药机制主要为23SrRNAA2143G突变。cagA基因与Hp对克拉霉素耐药不相关。     

Effect of Pretreatment Antibiotic Resistance to Metronidazole and Clarithromycin on Outcome of Helicobacter pylori Therapy

Our purpose was to define the effect of pretreatment Helicobacter pylori resistance to metronidazole or to clarithromycin on the success of antimicrobial therapy. We used 75 key words to perform a literature search in MEDLINE as well as manual searches to identify clinical treatment trials that provided results in relation to H. pylori susceptibility to metronidazole and clarithromycin or both during the period 1984–1997 (abstracts were not included). Meta-analysis was done with both fixed- and random-effect models; results were shown using Galbraith's radial plots. We identified 49 papers with 65 arms for metronidazole (3594 patients, 2434 harboring H. pylori strains sensitive to metronidazole and 1160 harboring resistant strains). Metronidazole resistance reduced effectiveness by an average of 37.7% (95% CI = 29.6–45.7%). The variability in the risk difference for metronidazole was 122.0 to –90.6 and the chi-square value for heterogeneity was significant (P < 0.001). Susceptibility tests for clarithromycin were performed in 12 studies (501 patients, 468 harboring H. pylori strains sensitive to clarithromycin and 33 harboring resistant strains). Clarithromycin resistance reduced effectiveness by an average of 55% (95% CI = 33–78%). We found no common factors that allowed patients to be divided into subgroups with additional factors significantly associated with resistance. In conclusion, metronidazole or clarithromycin pretreatment resistant H. pylori are the main factors responsible for treatment failure with regimens using these compounds. If H. pylori antibiotic resistance continues to increase, pretherapy antibiotic sensitivity testing might become necessary in many regions.     

Clarithromycin resistance and point mutations in the 23S rRNA gene in Helicobacter pylori isolates from Malaysia

OBJECTIVE: To determine the prevalence of primary clarithromycin resistance amongst Helicobacter pylori (H. pylori) strains in Malaysian patients with gastroduodenal diseases, by using restriction fragment length polymorphism (RFLP) in domain V of 23S rRNA. METHODS: Gastric biopsies were obtained from H. pylori positive patients undergoing gastroscopy. DNA extraction was followed by PCR amplification using the primers Hp23-1 and Hp23-2 flanking a region of 425bp within the bacterial 23S rRNA peptidyltranferase (Hp23S fragment). Analysis of the 23S rRNA gene mutations is based on the generation of restriction sites for two restriction enzymes: BbsI and BsaI, which correspond to the base substitutions characteristic of clarithromycin resistance from A to G at positions 2142 and 2143, respectively. RESULTS: Gastric biopsy samples were obtained from 107 patients. A fragment of size 425bp corresponding to that expected from amplification of domain V of 23S rRNA was PCR-amplified from only 105 samples. The amplicon was subsequently subjected to restriction by BbsI and BsaI. Only 1 sample (0.95%) had the BbsI mutation (base substitution at A2142G) and 2 samples (1.90%) the BsaI mutation (base substitution at A2143G). Thus 3 of 105 (2.9%) samples harbored clarithromycin resistant strains. CONCLUSION: In our experience, PCR-RFLP is a rapid and precise method to detect the resistance of H. pylori to clarithromycin. Using this method, a low prevalence of clarithromycin resistance was detected in our local Malaysian strains. This augurs well for the continued use of clarithromycin as a first line drug in the treatment and eradication of H. pylori infection.     

Prevalence of Resistance to Clarithromycin and Its Clinical Impact on the Efficacy of Helicobacter pylori Eradication

Background: Triple therapy with a proton-pump inhibitor (PPI) in combination with metronidazole and clarithromycin is the method of choice for eradication of Helicobacter pylori. Failures have been primarily blamed on the development of resistance to clarithromycin. The present study investigated the prevalence and clinical significance of resistance to clarithromycin and metronidazole in determining therapeutic success of both triple therapy as a primary eradication method and high-dose dual therapy in non-responders. Methods: On the basis of prior therapy, H. pylori-positive patients were assigned to one of two groups in the present prospective study. Group A (n = 93) included patients who had not undergone any prior eradication treatment, whereas group B (n = 15) consisted of patients who had received clarithromycin but in whom eradication had been unsuccessful. All patients underwent endoscopy with biopsy for bacterial culture and resistance studies. Patients in group A were treated with a 7-day regimen of pantoprazole (40 mg twice daily), metronidazole (500 mg twice daily), and clarithromycin (250 mg twice daily), whereas those in group B received omeprazole (40 mg three times a day) and amoxycillin (1000 mg three times a day ) for 14 days. Success of the eradication treatment was ascertained by means of the 13C urea breath test. Results: In group A resistance to clarithromycin and metronidazole was identified in 3 patients (4.9%) and in 14 patients (22.9%), respectively. Eradication proved successful in 78 of 84 patients (92.6%) followed up. Two of the 3 patients with primary clarithromycin resistance and 1 of the 14 patients with metronidazole resistance did not respond to treatment. In group B isolated or combined resistance to clarithromycin was found in seven patients, whereas another four showed isolated resistance to metronidazole. Eradication proved successful in 10 of 13 controlled patients (76.9%) followed up, and only 2 patients reported severe side effects. Conclusion: Determination of antibiotic resistance before initiating therapy is not necessary, since primary resistance to clarithromycin is rare. The Italian triple therapy remains a highly effective primary therapeutic method. Further, routine determination of resistance in non-responders also seems to be superfluous because high-dose dual therapy is an effective and well-tolerated second-line therapy regardless of the patients' resistance status.     

Clarithromycin resistance in Helicobacter pylori and its clinical relevance

SubjectheadingsHelicobacterpylon;Helicobacterinfections;clarithromycinresistanceINTRODUCTIONThemacrolideclarithromycinhasemergedasthemostimportantantibioticincombinedtherapyforeradicationofH.Pyloninfection[1'2).HOwever,concernsaboutincreasingclarithromycinresistanceinH.PylonanditsimpactontheefficacyoferadicationtherapyhavebeenraisedsinceitswidespreadacceptanceinH.Pylontherapy""'.Here,wesoughttoreviewthegeographicprevalenceofclarithromycinresistanceinH.Pylonanditsmolecularmechanisms,an…     

幽门螺杆菌克拉霉素异质性耐药及其特征分析

目的 对幽门螺杆菌(Helicobacter pylori, HP)克拉霉素异质性耐药进行检测,并分析异质性耐药的遗传进化特征。方法 对中国某医院50个¹³C尿素呼气试验阳性患者的胃窦黏液标本进行HP分离培养,每个样本挑取16个HP单克隆(分离培养平板中不满16个克隆者选取全部HP克隆),用Etest法测定各克隆的克拉霉素最低抑菌浓度(minimum inhibitory concentration, MIC)。进一步在异质性克拉霉素耐药病例中选取6份样本(MIC值差异较大的样本)相关的81个幽门螺杆菌克隆,利用多位点序列(MLST)分析的方法进行初步进化分析。结果 44个分离培养阳性的样本中24个全敏感,9个全耐药,11个为异质性耐药;6个MIC值差异较大的样本,最大值与最小值差别在100~1 000倍间。每个样本的9~16个克隆可分为1~6个ST型,结合vacA基因差异,分为2~8个基因型。遗传进化树显示2个样本的敏感、耐药克隆分为两簇,余4个样本中ST型与耐药间无明显关联。结论 HP在中国感染存在明显的克拉霉素异质性耐药现象,传统药敏试验可能不足以充分反映克拉霉素异质性耐药的现象,可能会引起误判,应高度关注。     

幽门螺杆菌基因多态性与克拉霉素耐药性关系的研究

目的研究幽门螺杆菌（Helicobacter pylori,Hp）对克拉霉素耐药情况及与23S rRNA基因点突变的关系。方法因上消化道症状进行胃镜检查的189例患者获得胃活检组织,微需氧培养得到坳,提取11例敏感菌和和19例耐药菌的DNA,对23S rRNA基因进行PCR扩增,再对敏感菌和耐药菌的23S rRNA基因进行全基因测序对比和生物信息学分析。结果Hp菌株对克拉霉素的耐药率是29．2％;19个对克拉霉素耐药的却菌株中17株出现23S rRNA基因突变,各种突变的比例分别为A→G36．8％、G→A21．5％、C→T15．8％、A→C10．5％和T→C5．3％。11例敏感株及2例耐药株均未发现23S rRNA基因突变。结论克拉霉素耐药的却菌株比较常见,23SrRNA基因的多个不同位点突变均与跏对克拉霉素耐药有关,而A—G和G—A突变是主要的形式。     

Helicobacter pylori Prevalence in Acquired Immunodeficiency Syndrome

Helicobacter pylori is consistently reported with high prevalence in HIV-negative patients with chronic gastritis and active ulcer disease. This study is an evaluation of the prevalence of H. pylori in AIDS patients, and the association with chronic gastritis, erosions, and ulcer disease. Seventy-three AIDS patients referred for the evaluation of gastrointestinal symptoms underwent upper endoscopy and antral gastric biopsy. Histologic gastritis was diagnosed and degree of activity graded on hematoxylin-eosin stain. H. pylori organisms were identified by acridine orange stain. A single pathologist evaluated the biopsy specimens. H. pylori was found in 15% (11 of 73) of AIDS patients. Histologic chronic active gastritis was evident in 94.5% (69 of 73) of the study group. H. pylori was identified in 15.9% (11 of 69) of biopsy specimens with histologic chronic active gastritis. The organism was more common in biopsy specimens with a higher grade of activity in the chronic-gastritis. Endoscopic erosions or ulcers were noted in 11 patients (seven gastric, four duodenal). H. pylori was present in 18% (2 of 11) of AIDS patients with erosions or ulcers. The prevalence of H. pylori in AIDS patients with histologic chronic active gastritis is much lower than the prevalence previously reported for HIV-negative patients with similar pathology. The low prevalence observed does not implicate H. pylori as the causal agent in most chronic active gastritis in the AIDS population. Impaired acid secretion may reduce colonization of gastric mucosa and explain the low rate of H. pylori observed.     

幽门螺杆菌临床菌株克拉霉素耐药性及耐药基因突变位点分析

摘要：目的：了解贵阳地区幽门螺杆菌（Helicobacter pylori,Hp）临床菌株对克拉霉素耐药性及克拉霉素耐药相关基因突变情况,为耐药性的快速检测提供依据。方法：采用琼脂稀释法对临床分离鉴定的Hp菌株,进行体外抗生素敏感试验,了解贵阳地区Hp临床株对克拉霉素耐药状况。选取Hp克拉霉素耐药的临床菌株10株、克拉霉素敏感的临床菌株4株和质控菌株2株,进行23S rRNA基因功能区V区片段的PCR扩增和测序,与GenBank中公布的Hp菌株相关序列进行比对分析。结果：贵阳地区Hp临床分离株对克拉霉素的耐药率达30.9%。贵阳地区10株Hp耐药菌的23S rRNA基因片段的碱基突变包括T2183C（10/10）、T2245C（9/10）、 A2144G（6/10）、C2196G（1/10）、A2204G（1/10）,4株敏感菌株在2183、2245、2196和2204位点也存在碱基差异,2144位点的基因突变仅存于耐药菌株中。结论：贵阳地区Hp克拉霉素耐药率较高,耐药菌株23SrDNA与耐药性相关的基因突变主要为A2144G。     

Transfer of Clarithromycin to Gastric Juice is Enhanced by Omeprazole in Helicobacter pylori -Infected Individuals

Background: The effects of proton-pump inhibitors and Helicobacter pylori infection on the distribution of drugs employed for the eradication of H. pylori are poorly understood. The aim of this study was to investigate the effects of a 7-day oral administration of 20 mg omeprazole on the distribution of clarithromycin in the gastric juice of individuals with H. pylori infection. Methods: Eighteen H. pylori infected dyspeptic male volunteers without endoscopic lesions were enrolled in a study with an open, randomized, two-period crossover design and a 21-day washout period between phases. Plasma and gastric juice concentrations of clarithromycin in subjects with and without omeprazole pretreatment were measured by means of liquid chromatography coupled to tandem mass spectrometry. Results: The maximum concentration of clarithromycin (C max ) and the area under the time-concentration curve from 0 to 2 h (AUC 0-2h ) were significantly higher in gastric juice than in plasma. Omeprazole treatment further augmented clarithromycin C max and AUC 0-2h in gastric juice approximately 2-fold ( P < 0.05). Conclusions: Short-term treatment with omeprazole in H. pylori -positive volunteers increases the amount of clarithromycin transferred to the gastric juice, confirming a synergism between these drugs. Our results suggest the presence of an active transport mechanism for clarithromycin from plasma to the gastric lumen, which is influenced by omeprazole.