Background: 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. The eradication rate of the the first line treatment has been declining in recent years and studies show first line treatment failure 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. Clarithromycin, metronidazole, levofloxacin, amoxicillin, and tetracycline are widely used in H. pylori eradication. Point mutations at 23S rRNA, gyrase A were reported to be associated with clarithromycin and levofloxacin resistance respectively. Our group found that the point mutations on 23S rRNA and gyraseA 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. However, molecular diagnosis of antimicrobial resistance against other antibiotics, such as metronidazole, amoxicillin and tetracycline still needs further investigations. 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. However, the composition of fecal samples was complex and it may cause a loss of sensitivity of test and detecting resistance against other antibiotics by fecal specimen was not reported. 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. We successfully detected mutations at 23S rRNA, and gyrA by nuclieic acid mass spectrometry in our preliminary work [NSC-103-2314-B-002-174] in 2014. We have successfully designed primers and probes to detect mutations among these genes in multiplex. We established 17 mutations of 23S rRNA, gyrA, 16S rRNA in two reactions. In addition, in order to confirm the infection of H. pylori, several specific genes including cagA and vacA were also designed. The detection limitation of this platform was about 10 copies and that was evaluated by serial dilution of H. pylori DNA. Hypothesis: It is possible to detect antimicrobial resistance of H. pylori by detecting genetic mutations. Nucleic acid mass spectrometry could detect antimicrobial resistance regardless of sample origin and quality. Aims: we want to find and validate genetic mutations associated with H. pylori resistance against metronidazole, amoxicillin, and tetracycline. We will use nucleic acid mass spectrometry to detect the resistance of H. pylori against amoxicillin, tetracycline and metronidazole in various clinical 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 3rd line H. pylori eradication. Material and methods: This project is composed of two parts: Part 1. Based on our study (103-2314-B-002-174) in 2014, we will sequence 16S rRNA, RdxA, FrxA, FrxB, pbp1A, hpB, hpC, omp25, omp3 gene and find out the mutations associated with resistance against metronidazole, amoxicillin and tetracycline in H. pylori isolates. We will further validate the consistency between genotypic resistance and phenotypic resistance in 100 gastric tissue specimen and 100 fecal specimen obtained from H. pylori infected hosts by nucleic acid mass spectrometry. Part 2. 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.