Analysis of Helicobacter pylori genotypes in clinical gastric wash samples
Helicobacter pylori is a key factor in the development of gastric cancer; indeed, clearance of H. pylori helps prevent gastric cancer. However, the relationship between gastric cancer and the abundance and diversity of H. pylori genotypes in the stomach remains unknown. Here, we present, for the first time, a quantitative analysis of H. pylori genotypes in gastric washes. A method was first developed to assess diversity and abundance by pyrosequencing and analysis of single nucleotide polymorphisms in 23S ribosomal RNA (rRNA), a gene associated with clarithromycin resistance. This method was then validated using arbitrarily mixed plasmids carrying 23S rRNA with single nucleotide polymorphisms. Multiple strains were detected in many of 34 clinical samples, with frequency 24.3 ± 24.2 and 26.3 ± 33.8 % for the A2143G and A2144G strains, respectively. Importantly, results obtained from gastric washes were similar to those obtained from biopsy samples. The method provides opportunities to investigate drug resistance in H. pylori and assess potential biomarkers of gastric cancer risk, and should thus be validated in large-scale clinical trials.
KeywordsHelicobacter pylori Gastric wash 23S rRNA Pyrosequencing
This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan to Y.W. (#25460937).
S.M., Y.W., and H.Y. conceived the study, designed and executed experiments, analyzed data, prepared figures and tables, and wrote the manuscript. S.O. analyzed data, and K.M. helped write the manuscript. R.O. and S.M. executed experiments and analyzed data. S.O. and K.M. obtained clinical samples. M.K. designed experiments, analyzed data, and helped write the manuscript. F.I. provided intellectual support. T.K., M.K., and N.S. supervised all aspects of the study.
Compliance with ethical standards
The study was conducted in accordance with all rules and regulations of the Hokkaido University Institutional Review Board (#014-0459), and informed consent was obtained from each patient.
Conflicts of interest
- 22.Fukase K, Kato M, Kikuchi S, Inoue K, Uemura N, Okamoto S, et al. Effect of eradication of Helicobacter pylori on incidence of metachronous gastric carcinoma after endoscopic resection of early gastric cancer: an open-label, randomised controlled trial. Lancet. 2008;372:392–7.CrossRefPubMedGoogle Scholar
- 29.Kawai T, Takahashi S, Suzuki H, Sasaki H, Nagahara A, Asaoka D, et al. Changes in the first line Helicobacter pylori eradication rates using the triple therapy—a multicenter study in the Tokyo metropolitan area (Tokyo Helicobacter pylori study group). J Gastroenterol Hepatol. 2014;29 Suppl 4:29–32.CrossRefPubMedGoogle Scholar