2014-08-012024-05-18https://scholars.lib.ntu.edu.tw/handle/123456789/710834Background: Helicobacter pylori (H. pylori) is an important causal factor for gastric cancer, peptic ulcer disease, and MALT-type lymphoma. Eradication of H. pylori prevents gastric cancer and decrease incidence of peptic ulcer disease. However, the eradication rate of the widely used clarithromycin-containing triple therapy in first line treatment has been declining in recent years. Studies show that eradication rate of triple therapy is closely related to H. pylori resistance to antibiotics. For patient with refractory H. pylori infection, the Maastricht III consensus recommended to choose antibiotics according to drug susceptibility test. However, there were some limitations of the traditional susceptibility test: requiring endoscopy examination, time consuming, and relatively low yield rate. Recently, point mutations at 23S rRNA, gyrase A, RdxA and 16S rRNA were reported to be associated with clarithromycin, levofloxacin, metronidazole and tetracycline resistance, respectively. Our group found that the point mutations on 23S rRNA and gyrase A gene correlated with treatment failure. There is a high consistence between the genotypic resistance determined by gastric tissue or H. pylori isolates and phenotypic resistance. The eradication rate of genotypic resistance guided treatment was 83%. Fecal samples has advantages on non-invasiness and easy to be obtained. Studies have showed it is possible to detect clarithromycin resistance by nested PCR in fecal samples, but detecting resistance to other antibiotics was not reported. Literature suggested that the quality of fecal samples or the DNA extract may cause a loss of sensitivity of test. Whether RNAlater or S.T.A.R buffer could be used to preserve fecal samples for further antibiotic resistance screening is not known. Nucleic acid mass spectrometry is a multiple function platform with high sensitivity and specificity. We have successfully utilized it for multiplex gene testing in cancer and for drug resistance related gene mutation detection in Mycobacterium tuberculosis. We can detect totally 45 mutation sites among 8 genes at the same time in 1 to 2 reactions. The detection limitation can be less than 10 copies. Therefore, this platform is suitable to develop the molecular diagnostics assay in specimens with poor quality and complex mixture. Hypothesis: the quality of stored fecal samples will decrease the sensitivity of genotypicl resistance detection and RNAlater or S.T.A.R buffer could be used to preserve fecal samples to improve quality of samples for further molecular testing. Nucleic acid mass spectrometry could detect antimicrobial resistance in fecal samples regardless of sample quality. Aims: we want to evaluate whether RNAlater or S.T.A.R buffer could be used in pre-analytic fecal samples storage. We will use nucleic acid mass spectrometry to directly detect the resistance of H. pylori against clarithromycin, levofloxacin, tetracycline and metronidazole in stool specimens and will evaluate its accuracy. We will also use this platform to study secondary antibiotic resistance of H. pylori and conduct a genotypic resistance tailored treatment for 3 rd line H. pylori eradication. Material and methods: This project is composed of three parts: Part 1. The correlation between genotypic resistance and phenotypic resistance will be evaluated in 50 H. pylori isolates. The genotypic resistance will be determined by nucleic acid mass spectrometry and the phenotypic resistance will be determined by agar dilution tests. We will then utilize nucleic acid mass spectrometry to detect the resistance of H. pylori against clarithromycin, levofloxacin, tetracycline and metronidazole in 200 fresh stool samples. Part 2. Fecal samples will be stored in different conditions or pretreated by different kind of buffer solutions. We will compare the consistency of genotypic resistance result between stored and fresh fecal samples by nucleic acid mass spectrometry and direct sequencing. Part 3. We will use this platform to study secondary resistance after H. pylori infection and investigate the possible risk factor associated with secondary resistance. Furthermore, we will conduct a third line H. pylori eradication trial guided by this non-invasive antimicrobial resistance test.Helicobacter pylorinucleic acid mass spectrometryantimicrobial resistancemolecular diagnosis