從胃液快速診斷幽門螺旋桿菌及其clarithromycin抗藥性與宿主CYP2C19基因多型性

Rapid diagnosis of Helicobacter pylori and its clarithromycin resistance and CYP2C19 polymorphism of host from gastric juice

楊瀞嘉(Ching-Chia Yang)

指導教授 ： 張玲麗

共同指導教授 ： 吳登強(Deng-Chyang Wu)

高雄醫學大學/醫學院/醫學研究所/碩士(2013年)

https://doi.org/10.6832/KMU.2013.00062

摘要

幽門螺旋桿菌會導致胃部疾病發生甚至有致癌的危險，但成功的使用抗生素治療可以使胃潰瘍、十二指腸潰瘍，甚至惡性的胃黏膜相關性淋巴瘤痊癒或降低再發生率。目前國際性治療準則所建議的幽門螺旋桿菌第一線根除治療用藥為標準三合療法，包括一種氫離子阻斷劑（PPIs）加上兩種抗生素，如clarithromycin和amoxicillin（或metronidazole），但近年來標準三合療法的成功率逐漸下降，國內外之成功率不到80%，主要失敗原因為病人對藥物之順從性不良與細菌對抗生素產生抗藥性之外，還有宿主之基因背景如CYP2C19之基因多型性被認為會影響氫離子阻斷劑之作用，因而影響治療之成功率。細菌抗藥性逐年不斷增加，特別是clarithromycin抗藥性菌株難以根除，並且為標準三合療法主要失敗之因素。此外，傳統診斷細菌對藥物之感受性需先行細菌培養，之後再以E-test偵測細菌對各種抗生素之感受性，需耗時至少一星期，臨床醫師才知道是否正確給藥，並且若需考慮CYP2C19之基因多型性，還必須另外再收集血液檢體進行限制酶片段長度多型性聚合酶鏈鎖反應分析。基於尋求快速且成功給藥治療幽門螺旋桿菌感染之方法，本研究藉由胃鏡檢查時收集6ml病患胃液，並藉著萃取可能存在於胃液之幽門螺旋桿菌及脫落之宿主胃上皮細胞的DNA，再以聚合酶鏈鎖反應擴增幽門螺旋桿菌urease A與cag A基因，以判定是否有幽門螺旋桿菌感染，並以限制酶片段長度多型性聚合酶鏈鎖反應增幅幽門螺旋桿菌clarithromycin抗藥基因23S rRNA及宿主CYP2C19基因，再利用酵素Mbo II和Bsa I分別切割23S rRNA以偵測A2142G和A2143G突變，以及以酵素Sma I和Bam HI切割CYP2C19 m1, m2基因，以得知細菌是否對clarithromycin具抗藥性，以及CYP2C19之基因表現型為快速代謝型（extensive metabolizer, EM）或緩慢代謝型（poor metabolizer, PM）。本研究結果顯示，胃液檢體檢測幽門螺旋桿菌感染的敏感性為92.1%（105/114），特異性亦為92.9%（143/154），陽性預測值為90.5%（105/116），陰性預測值為94.1%（143/152）及準確度為92.5%（248/268）。另外，亦可以從胃液檢體檢測到幽門螺旋桿菌之23S rRNA基因，並分析clarithromycin抗藥性，敏感性為97.3%（36/37），特異性為91.5%（43/47），陽性預測值為90.0%（36/40），陰性預測值為97.7%（43/44）及準確度為94.0%（79/84），檢測出有clarithromycin抗藥性的病患大多為23S rRNA基因之A2143G的突變，只有一位病患為A2142G的突變。除此之外，由胃液或血液檢體分析CYP2C19基因多型性之一致性高達94.9%（149/157）。因此，利用胃液檢體以聚合酶鏈鎖反應或限制酶片段長度多型性聚合酶鏈鎖反應診斷幽門螺旋桿菌感染及其抗藥性與宿主CYP2C19基因多型性，不僅實驗流程簡單，此外由於此方法不需透過培養幽門螺旋桿菌進行E-test抗生素感受性試驗，亦不需額外採集血液檢體，因此可將檢驗時間縮短為2天，並達到高敏感度、特異性、陽性預測值、陰性預測值及準確度，更快速提供醫師如何選用適合的抗生素以治療幽門螺旋桿菌感染，還可以提供醫師得知病患所屬的藥物代謝CYP2C19基因多型性，以判定病患對藥物代謝的速率，更快速提供臨床醫師正確之藥物治療以清除幽門螺旋桿菌，以提高根除幽門螺旋桿菌成功率。

關鍵字

胃液 ； 幽門螺旋桿菌 ； clarithromycin抗藥性 ； CYP2C19基因多型性 ； 聚合酶鏈鎖反應 ； 限制酶片段長度多型性聚合酶鏈鎖反應

並列摘要

In spite of the risk of carcinogenesis of Helicobacter pylori (H. pylori, Hp), appropriate antibiotics usage can decrease the development of gastric ulcer、duodenal ulcer、and malignant mucosa associated lymphoma. Triple therapy with a proton pump inhibitor (PPI), clarithromycin and amoxicillin (or metronidazole) is considered the standard first-line regimen for eradication of H. pylori. Even with the currently most effective treatment regimens, about 20% of patients fail to eradicate H. pylori infection. Many factors affect the efficacy of eradication, such as poor patient compliance and antimicrobial resistance. Besides these, the host genetic background, such as CYP2C19 polymorphism status was considered to determine the outcome of PPI-based triple therapy. Bacterial resistance increasing year by year, especially clarithromycin resistant strains are difficult to eradicate and is a major factor of triple therapy failure. Traditionally, bacterial must be cultured first, and then susceptibility to antimicrobial agents was determined by E-test. Totally, it nearly spends one week for the physician to realize whether properly antimicrobial agents was used to treat the disease. It also should be blood sampling to determine the CYP2C19 gene polymorphism by polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP). In this study, in order to treat H. pylori infection successfully, 6 ml specimens of gastric juice were collected from patients while receive gastroscopy. DNA was extracted from gastric juice and then urease A and cag A was amplified by polymerase chain reaction (PCR) for detecting the existence of H. pylori. By PCR-RFLP, the 23S rRNA and CYP2C19 genes of H. pylori and host, respectively were amplified and restriction enzymes Mbo II and Bsa I were used to detect 23S rRNA A2142G and A2143G mutations; Sma I and Bam HI were used to analyze CYP2C19 gene polymorphism in order to realize the sensitivity of clarithromycin and CYP2C19 polymorphism. Our results showed that the sensitivity (SEN), specificity (SPE), positive predictive value (PPV), negative predictive value (NPV) and accuracy (ACC) to detect H. pylori infection from gastric juice by PCR amplification of urease A/cag A was 92.1%（105/114）, 92.9%（143/154）, 90.5%（105/116）, 94.1%（143/152） and 92.5%（248/268）, respectively. The SEN, SPE, PPV and NPV to detect clarithromycin resistance from gastric juice were 97.3% (36/37), 91.5% (43/47), 90.0% (36/40) and 97.7% (43/44), respectively. In addition, PCR-RFLP of 23S rRNA from gastric juice is 94.0% (79/84) consistence with traditional E-test to detect clarithromycin resistance. Most of clarithromycin resistance is due to A2143G mutation in 23S rRNA, and only one of clarithromycin resistance is due to A2142G mutation in 23S rRNA. By using PCR-RFLP, the consistence of human CYP2C19 gene polymorphism from human blood and gastric juice was as high as 94.9% (149/157). Due to unnecessary of bacterial culturing, E-test for antibiotic susceptibility and extra blood sampling, diagnosis can be shortened to two days. Other advantages include simple experimental procedure and can achieve high SEN, SPE, PPV, NPV and ACC. Therefore, manipulate gastric juice actually be a more effective diagnostic samples for evaluation H. pylori existence, clarithromycin resistance, and host CYP2C19 gene polymorphism. It provides clinicians to give the correct medications to treat H. pylori infection as soon as possible and improve the eradication rate of H. pylori infection.

並列關鍵字

gastric juice ； Helicobacter pylori ； H. pylori ； Hp ； clarithromycin resistance ； CYP2C19 gene polymorphism ； polymerase chain reaction ； PCR ； polymerase chain reaction-restriction fragment length polymorphisms ； PCR-RFLP

參考文獻

1. 1994a. NIH Consensus Conference. Helicobacter pylori in peptic ulcer disease. NIH Consensus Development Panel on Helicobacter pylori in Peptic Ulcer Disease. JAMA : the journal of the American Medical Association 272, 65-69.

2. 1994b. Schistosomes, liver flukes and Helicobacter pylori. IARC Working Group on the Evaluation of Carcinogenic Risks to Humans. Lyon, 7-14 June 1994. IARC monographs on the evaluation of carcinogenic risks to humans / World Health Organization, International Agency for Research on Cancer 61, 1-241.

3. Abrante, L., Reyes, N., Garcia-Amado, M.A., Suarez, P., Romero, R., Michelangeli, F., Contreras, M., 2012. [Diagnosis of Helicobacter pylori infection by PCR in gastric juice and gastroesophageal biopsies from dyspeptic patients]. Investigacion clinica 53, 168-177.

4. Beswick, E.J., Suarez, G., Reyes, V.E., 2006. H pylori and host interactions that influence pathogenesis. World journal of gastroenterology : WJG 12, 5599-5605.

5. Chen, T.S., Chang, F.Y., Lee, S.D., 1995. A biopsy urease test in the detection of Helicobacter pylori: comparison of antral and body specimens. Zhonghua yi xue za zhi = Chinese medical journal; Free China ed 55, 361-365.