評估幽門螺旋桿菌感染的抗藥性檢測方式與第一線療法

Abstract

自從幽門螺旋桿菌被發現以來，陸續的研究證實它和多種重要的消化道疾病有關，這些疾病包括慢性胃炎、消化性潰瘍、胃癌及胃黏膜組織相關淋巴瘤等。最近15年，含有質子幫浦抑制劑 (proton pump inhibitor, PPI) 的三合一療法被視為清除幽門螺旋桿菌感染首選。但是隨著抗生素抗藥性的增加，目前許多地區三合一療法的有效率已低於80%，因此，依幽門螺旋桿菌的抗生素敏感性試驗結果做除菌，以避免治療失敗變得格外重要。此外，加拿大及歐洲最新治療指引建議clarithromycin高抗藥性 (>15-20%) 地區以含鉍鹽四合一療法取代現有一線治療。基於以上原因，本研究分為以下兩個部分進行基礎及臨床試驗。 第一部分，近年來，隨著幽門螺旋桿菌對clarithromycin抗藥性的產生，使得標準三合一療法的除菌率在全球有下降的趨勢。因此，在高clarithromycin抗藥性的區域，細菌培養 (bacteria culture) 及標準敏感性試驗 (standard susceptibility testing) 就變得相當重要。根據目前最新歐洲治療指引建議，在無法執行標準敏感性試驗時，可以分子診斷 (genotypic testing) 的方式來替代。但這樣的檢測方法是否能真實反應臨床治療上的結果仍須被證實。因此，本研究希望能進一步評估genotypic testing在臨床治療上所扮演的角色，提供臨床作為藥物治療最佳的選擇指標。本研究收錄250位以clarithromycin-containing triple therapy (CLA-TT) 治療幽門螺旋桿菌感染的病患，其中的150位來自之前多中心研究的病患，另外的100位則來自過去於門診治療過的病患，而這些病患的菌株均在給予治療前已採檢完畢。以PCR-RFLP的分子診斷方式來分析幽門螺旋桿菌23S rRNA基因V區的第2143及2142位置，是否由adenine被guanine取代 (即A2143G及A2142G)。我們使用E-test來檢測clarithromycin的MIC值，作為判定genotypic testing準確與否的標準，並定義MIC ≥1 μg/mL則其表現型呈抗藥性。然而，genotypic testing是否有足夠的可信度則藉由其與臨床治療結果的關係來判斷。以E-test檢測有16.8% (42/250) 呈抗藥性 (phenotypic resistant)，MIC值的範圍為 < 0.016至> 256 μg/mL。Phenotypic resistant及sensitive之病人除菌率分別為23.5% (10/42) 及92.8% (193/208)。Phenotypic resistant被治癒的10位病人中，有9位病人的MIC ≤ 6 μg/mL，即有90%的病人為low-grade resistance，這樣的結果表示MIC值與臨床治療效果有密切關係存在。另一方面，genotypic testing有12.4% (31/250) 呈抗藥性，且全部均為A2143G點突變的抗藥性菌株。即便phenotypic及genotypic testing的一致率高達91.6% (229/250)，但genotypic resistant的菌株其MIC值分布相當廣泛，這樣的結果指出，genotypic testing無法準確預測MIC值及抗藥性程度的高低。而在42位phenotypic resistant的病人當中，有16位 (38.1%) 以genotypic testing的方式檢測為偽陰性，16位當中有14位其MIC值 ≥ 24 μg/mL，這16位病人因呈偽陰性而給予治療，而導致9位 (56.3%) 病人治療失敗。208位病人當中有5位具有A2143G點突變，但這5位的MIC值均 ≤ 0.25 μg/mL，而且有4位治療成功。因此，我們認為以genotypic testing檢測幽門螺旋桿菌23S rRNA的A2143G或A2142G，並無法為使用CLA-TT治療的病人，提供一個令人滿意的臨床結果的預測，同時更無法取代standard susceptibility testing在臨床上的應用。 第二部分，隨著clarithromycin抗藥性的增加，在很多地區三合一療法的有效度已低於80%，而需要再找尋更好的治療方式。在歐洲，含有鉍鹽的四合一療法(BQT)目前已被推薦為高clarithromycin抗藥性 (>15-20%) 地區的第一線用藥。而另一方面，最近我們使用高劑量的二合一療法 (HDDT) 和三合一療法及接續性療法進行比較研究，結果顯示HDDT的幽門螺旋桿菌除菌率可以達到95%以上，明顯優於另外兩種療法。故本試驗的目的為：(1) 比較HDDT及BQT作為第一線治療的療效；(2) 比較服用這些療法的副作用及服藥順從性；(3) 探討可能影響這些療法的療效的因素，包括CYP2C19基因型及抗生素抗藥性等。這是一項國內多中心的研究。本研究將納入經內視鏡確診患有幽門螺旋桿菌感染引發胃炎、消化性潰瘍與十二指腸潰瘍之成年病人，進行隨機分派至HDDT (rabeprazole 20 mg與amoxicillin 750 mg，一天口服四次，共十四天) 或 BQT (rabeprazole 20 mg一天口服兩次，合併用tripotassium dicitrate bismuthate 300 mg與metronidazole 250 mg以及tetracycline 500 mg，一天口服四次，共十天)。服藥後以碳13呼氣試驗評估其療效。本研究自2015年7月至2016年4月試驗期間，每組規劃納入210位，實際共納入244位尚未接受過除菌治療的幽門螺旋桿菌感染陽性病人，所有病人以平均比例隨機分派至各組療法。HDDT之除菌率為91.0% (95%信賴區間：85.9-96.1%)，BQT之除菌率為89.3% (95%信賴區間：83.9-94.8％)；療效上，HDDT不劣於BQT (p=0.0321)，甚至比BQT還要好，但並沒有統計差異。但副作用發生率兩者具有統計差異 (HDDT: 14.6% vs. BQT: 42.7%, p<0.0001)，服藥順從性兩者無顯著差異。故結論為HDDT用於一線治療幽門螺旋桿菌感染與BQT一樣好，且副作用更少。

Helicobacter pylori (H. pylori) is strongly associated with chronic gastritis, peptic ulcers, gastric cancer and gastric MALT lymphoma. Currently, a combination therapy of a proton pump inhibitor (PPI) and antibiotics is used as the first-choice treatment for H. pylori infection. Eradication rates of triple therapy for H. pylori infection have been rapidly falling to below 80% in most area as a result of a progressive increase of resistance to clarithromycin (CLA). Thus, treatment should be guided by antimicrobial susceptibility testing to prevent treatment failure. In addition, Toronto and Maastricht IV Consensus suggest in area of high clarithromycin resistance, bismuth-containing quadruple therapy (BQT) is recommended for first-line empirical treatment. Based on above, our studies included two parts of results in different aspects of this issue. 【Part I】Resistance of H. pylori to CLA significantly reduces the efficacy of CLA-containing regimens. Thus, it is important to perform culture and standard susceptibility testing in a region with high prevalence of CLA-resistance (CLA-R). Recently, published guidelines suggest the use of genotypic testing as an alternative method if the standard susceptibility testing is not available. However, the reliability of genotypic testing in clinical practice still needs to be verified. We aimed to evaluate the role of genotypic testing in clinical practice. Genotypic testing for CLA-R was conducted in a group of 250 patients treated with CLA-containing triple therapy (CLA-TT) from a multicenter study. Genotypic testing was performed using PCR-RFLP in duplicate to analyze A2143G or A2142G mutations at 23S rRNA gene of H. pylori. The E-test was used to determine the minimal inhibitory concentrations (MICs) of CLA. The phenotypic resistance was defined as MIC ≥ 1 mg/L and was used as a standard to determine the accuracy of genotypic testing. Agar dilution method was performed to confirm the phenotypic resistance when discordance occurred. The reliability of genotypic testing in clinical practice was assessed by its correlation with therapeutic outcome. Using H. pylori culture with E-test, 42/250 (16.8%) isolates were identified to be CLA-R (phenotypic resistant) with MIC ranging from < 0.016 to > 256 μg/mL. The cure rates in phenotypic resistant and sensitive patients were 10/42 (23.8%) and 193/208 (92.8%), respectively. In phenotypic resistant patients who were cured, 9/10 (90%) of H. pylori strains had low-grade resistance (MIC ≤ 6 μg/mL), indicating MIC value correlated closely with the therapeutic outcome. On the other hand, according to genotypic testing, 31 (12.4%) isolates were CLA-R and all of them were identified with an A2143G point mutation. Although a concordance between the phenotypic and genotypic testing was present in 229 (91.6%) patients, the wide MIC distribution in genotypic resistant strains indicates the molecular test was unable to accurately predict its MIC value and the grade of resistance. In the 42 phenotypic resistant patients, genotyping was falsely negative in 16 (38.1%) patients, 14 of 16 had MIC of ≥ 24 μg/mL, leading to an unnecessary treatment failure in 9/16 (56.3%) of these patients. Five out of 208 phenotypic sensitive patients had the genetic mutation but had MIC ≤ 0.25 μg/mL (4 of 5 were cured). Current genotypic analysis at 23S rRNA for A2143G or A2142G mutations in H. pylori strains provides an unsatisfactory prediction on treatment outcome in CLA-TT and cannot replace standard susceptibility testing in clinical practice. 【Part II】The efficacy of CLA-TT for H. pylori infection has decreased below 80% because of increasing resistance to clarithromycin. In Europe, bismuth-containing quadruple therapy (BQT) consisting of a PPI, bismuth, metronidazole, and tetracycline for 10-14 days has been recommended for the standard first-line treatment in areas with prevalence of clarithromycin resistance > 15-20%. Recently, we have completed a large-scale randomized controlled trial. Our results show that high-dose dual therapy (HDDT) with rabeprazole 20 mg and amoxicillin 750 mg four times daily for 2 weeks cures more than 95% of treatment-naive patients and is superior to standard triple therapy or sequential therapy. Up to now, to our knowledge, there is few randomized, large scale study prospectively and simultaneously comparing the efficacy, adverse effects and patient adherence of HDDT and BQT as 1st-line regimens for H. pylori eradication in and out of our country. The aims of this study are (1) to compare the efficacy of HDDT, and BQT as 1st-line regimen in H. pylori eradication; (2) to compare the patient adherence and adverse effects of these treatment regimens; (3) to investigate factors that may influence H. pylori eradication by these treatment regimens. We performed a large-scale multihospital trial to compare the efficacy of HDDT and BQT in treatment-naive (n=244) patients with H. pylori infection. Treatment-naive patients were randomly assigned to groups given HDDT (rabeprazole 20 mg and amoxicillin 750 mg, qid for 14 days, group A) and BQT (rabeprazole 20 mg bid + tripotassium dicitrate bismuthate 300 mg qid + metronidazole 250 mg qid + tetracycline 500 mg qid for 10 days, group B). H. pylori infection was detected by using the 13C-urea breath test. We evaluated factors associated with treatment outcomes. In the intention-to-treat analysis, H. pylori was eradicated in 91.0% of patients in group A (95% CI, 85.9-96.1%) and 89.3% in B (95% CI, 83.9%-94.8%). The efficacy of HDDT was as good as BQT irrespective of CYP2C19 genotype and there was no significant difference between groups in patient adherence. However, there was significant difference between groups in adverse events. HDDT should be considered for first-line treatment in view of the rising prevalence of clarithromycin-resistant H pylori.