title: Quantitative Detection and Genotyping of Helicobacter pylori from Stool using Droplet Digital PCR Reveals Variation in Bacterial Loads that Correlates with cagA Virulence Gene Carriage.
abstract: BACKGROUND
  Epidemiologic studies of the carcinogenic stomach bacterium Helicobacter pylori have been limited by the lack of noninvasive detection and genotyping methods. We developed a new stool-based method for detection, quantification, and partial genotyping of H. pylori using droplet digital PCR (ddPCR), which allows for increased sensitivity and absolute quantification by PCR partitioning.
  
  
  METHODS
  Stool-based ddPCR assays for H. pylori 16S gene detection and cagA virulence gene typing were tested using a collection of 50 matched stool and serum samples from Costa Rican volunteers and 29 H. pylori stool antigen-tested stool samples collected at a US hospital.
  
  
  RESULTS
  The stool-based H. pylori 16S ddPCR assay had a sensitivity of 84% and 100% and a specificity of 100% and 71% compared to serology and stool antigen tests, respectively. The stool-based cagA genotyping assay detected cagA in 22 (88%) of 25 stools from CagA antibody-positive individuals and four (16%) of 25 stools from CagA antibody-negative individuals from Costa Rica. All 26 of these samples had a Western-type cagA allele. Presence of serum CagA antibodies was correlated with a significantly higher load of H. pylori in the stool.
  
  
  CONCLUSIONS
  The stool-based ddPCR assays are a sensitive, noninvasive method for detection, quantification, and partial genotyping of H. pylori. The quantitative nature of ddPCR-based H. pylori detection revealed significant variation in bacterial load among individuals that correlates with presence of the cagA virulence gene. These stool-based ddPCR assays will facilitate future population-based epidemiologic studies of this important human pathogen.
authors.affiliation: Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA, USA.
authors.forename: Sarah
authors.initials: S
authors.lastname: Talarico
authors.affiliation: National Cancer Institute, NIH, Rockville, MD, USA.
authors.forename: Mahboobeh
authors.initials: M
authors.lastname: Safaeian
authors.affiliation: Proyecto Epidemiológico Guanacaste, Fundación INCIENSA, Guanacaste, Costa Rica.|International Agency for Research on Cancer, Lyon, France.
authors.forename: Paula
authors.initials: P
authors.lastname: Gonzalez
authors.affiliation: National Cancer Institute, NIH, Rockville, MD, USA.
authors.forename: Allan
authors.initials: A
authors.lastname: Hildesheim
authors.affiliation: Proyecto Epidemiológico Guanacaste, Fundación INCIENSA, Guanacaste, Costa Rica.|International Agency for Research on Cancer, Lyon, France.
authors.forename: Rolando
authors.initials: R
authors.lastname: Herrero
authors.affiliation: Proyecto Epidemiológico Guanacaste, Fundación INCIENSA, Guanacaste, Costa Rica.
authors.forename: Carolina
authors.initials: C
authors.lastname: Porras
authors.affiliation: Proyecto Epidemiológico Guanacaste, Fundación INCIENSA, Guanacaste, Costa Rica.
authors.forename: Bernal
authors.initials: B
authors.lastname: Cortes
authors.affiliation: Clinical Microbiology Laboratory, Harborview Medical Center, Seattle, WA, USA.|Department of Laboratory Medicine, University of Washington, Seattle, WA, USA.
authors.forename: Ann
authors.initials: A
authors.lastname: Larson
authors.affiliation: Clinical Microbiology Laboratory, Harborview Medical Center, Seattle, WA, USA.|Department of Laboratory Medicine, University of Washington, Seattle, WA, USA.|Department of Microbiology, University of Washington, Seattle, WA, USA.
authors.forename: Ferric C
authors.initials: FC
authors.lastname: Fang
authors.affiliation: Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA, USA.|Department of Microbiology, University of Washington, Seattle, WA, USA.
authors.forename: Nina R
authors.initials: NR
authors.lastname: Salama
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000942:Antigens, Bacterial / Q000032:analysis* / Q000235:genetics; D058491:Bacterial Load / Q000379:methods*; D001426:Bacterial Proteins / Q000032:analysis* / Q000235:genetics; D002648:Child; D002675:Child, Preschool; D003364:Costa Rica; D005243:Feces / Q000382:microbiology*; D005260:Female; D060005:Genotyping Techniques / Q000379:methods; D016481:Helicobacter Infections / Q000382:microbiology*; D016480:Helicobacter pylori / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D007223:Infant; D008297:Male; D008875:Middle Aged; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012680:Sensitivity and Specificity; D014481:United States; D037521:Virulence Factors / Q000032:analysis* / Q000235:genetics; D055815:Young Adult
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D012336:RNA, Ribosomal, 16S; D037521:Virulence Factors; C080866:cagA protein, Helicobacter pylori
keywords: Droplet digital PCR; H. pylori; bacterial load; cagA gene; stool-based assay