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

• Main Authors: Ching-Chia Yang, 楊瀞嘉
• Other Authors: Lin-Li Chang
• Format: Others
• Language: zh-TW
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/56136423720272131992

碩士 === 高雄醫學大學 === 醫學研究所 === 101 === 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.