title: An Appropriate Cutoff Value for Determining the Colonization of Helicobacter pylori by the Pyrosequencing Method: Comparison with Conventional Methods.
abstract: BACKGROUND
  Sequencing of 16S ribosomal RNA (rRNA) gene has improved the characterization of microbial communities. It enabled the detection of low abundance gastric Helicobacter pylori sequences even in subjects that were found to be H. pylori negative with conventional methods. The objective of this study was to obtain a cutoff value for H. pylori colonization in gastric mucosa samples by pyrosequencing method.
  
  
  METHODS
  Gastric mucosal biopsies were taken from 63 subjects whose H. pylori status was determined by a combination of serology, rapid urease test, culture, and histology. Microbial DNA from mucosal samples was amplified by PCR using universal bacterial primers. 16S rDNA amplicons were pyrosequenced. ROC curve analysis was performed to determine the cutoff value for H. pylori colonization by pyrosequencing. In addition, temporal changes in the stomach microbiota were observed in eight initially H. pylori-positive and eight H. pylori-negative subjects at a single time point 1-8 years later.
  
  
  RESULTS
  Of the 63 subjects, the presence of H. pylori sequences was detected in all (28/28) conventionally H. pylori-positive samples and in 60% (21/35) of H. pylori-negative samples. The average percent of H. pylori reads in each sample was 0.67 ± 1.09% in the H. pylori-negative group. Cutoff value for clinically positive H. pylori status was approximately 1.22% based on ROC curve analysis (AUC = 0.957; p < .001). Helicobacter pylori was successfully eradicated in five of seven treated H. pylori-positive subjects (71.4%), and the percentage of H. pylori reads in these five subjects dropped from 1.3-95.18% to 0-0.16% after eradication.
  
  
  CONCLUSIONS
  These results suggest that the cutoff value of H. pylori sequence percentage for H. pylori colonization by pyrosequencing could be set at approximately 1%. It might be helpful to analyze gastric microbiota related to H. pylori sequence status.
authors: Department of Internal Medicine, Seoul National University Bundang Hospital, Seongnam, Gyeonggi-do, South Korea.|Department of Internal Medicine, Liver Research Institute, Seoul National University College of Medicine, Seoul, South Korea.
authors: Jaeyeon
authors: J
authors: Kim
authors: Department of Internal Medicine, Seoul National University Bundang Hospital, Seongnam, Gyeonggi-do, South Korea.|Department of Internal Medicine, Liver Research Institute, Seoul National University College of Medicine, Seoul, South Korea.
authors: Nayoung
authors: N
authors: Kim
authors: Department of Internal Medicine, Seoul National University Bundang Hospital, Seongnam, Gyeonggi-do, South Korea.
authors: Hyun Jin
authors: HJ
authors: Jo
authors: Department of Internal Medicine, Liver Research Institute, Seoul National University College of Medicine, Seoul, South Korea.
authors: Ji Hyun
authors: JH
authors: Park
authors: Department of Internal Medicine, Seoul National University Bundang Hospital, Seongnam, Gyeonggi-do, South Korea.
authors: Ryoung Hee
authors: RH
authors: Nam
authors: Department of Biological Sciences, Institute of Microbiology, Seoul National University, Seoul, South Korea.
authors: Yeong-Jae
authors: YJ
authors: Seok
authors: Department of Biological Sciences, Institute of Microbiology, Seoul National University, Seoul, South Korea.
authors: Yeon-Ran
authors: YR
authors: Kim
authors: Department of Internal Medicine, Liver Research Institute, Seoul National University College of Medicine, Seoul, South Korea.
authors: Joo Sung
authors: JS
authors: Kim
authors: Department of Microbiology, College of Medicine, Hanyang University, Seoul, South Korea.
authors: Jung Mogg
authors: JM
authors: Kim
authors: NAR Center, Inc., Daejeon Oriental Hospital of Daejeon University, Daejeon, South Korea.
authors: Jung Min
authors: JM
authors: Kim
authors: Department of Internal Medicine, Seoul National University Bundang Hospital, Seongnam, Gyeonggi-do, South Korea.|Department of Internal Medicine, Liver Research Institute, Seoul National University College of Medicine, Seoul, South Korea.
authors: Dong Ho
authors: DH
authors: Lee
authors: Department of Internal Medicine, Liver Research Institute, Seoul National University College of Medicine, Seoul, South Korea.
authors: Hyun Chae
authors: HC
authors: Jung
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D058491:Bacterial Load; D001706:Biopsy; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D005260:Female; D005753:Gastric Mucosa / Q000382:microbiology*; D016481:Helicobacter Infections / Q000175:diagnosis* / Q000382:microbiology; D016480:Helicobacter pylori / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D008297:Male; D008875:Middle Aged; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S
keywords: 16S ribosomal RNA; Helicobacter pylori; gastric microbiota; stomach