title: Polymerase chain reaction--restriction fragment length polymorphism analysis of clarithromycin-resistant Helicobacter pylori infection in children using stool sample.
abstract: BACKGROUND
  To analyze clarithromycin-resistant Helicobacter pylori infection in children, we developed a method of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis using stool samples.
  
  
  METHODS
  Twenty-three children without significant upper abdominal symptoms were included (mean age 7.0 years). Of these, 18 and five were diagnosed as H. pylori-positive and -negative, respectively, by the H. pylori stool antigen test (HpSA). The DNA from the stool samples was purified using the QIAamp DNA Stool Minikit (QIAGEN). The PCR was performed on the purified DNA using oligonucleotide primers designed to amplify the 23S rRNA gene of H. pylori. The PCR products were reacted with restriction enzymes MboII, BceAI, and BsaI to detect mutations A2142G, A2142C, and A2143G, respectively.
  
  
  RESULTS
  Sixteen of the 18 HpSA-positive samples were PCR-positive, and all five HpSA-negative samples were PCR-negative. Thus, the PCR had 89% sensitivity and 100% specificity, with 91% accuracy in reference to HpSA. Of the 16 PCR-positive samples, one and four were digested with MboII and BsaI, respectively, indicating 31% prevalence of CAM-resistance.
  
  
  CONCLUSIONS
  We conclude that the PCR-RFLP using stool samples is a rapid and reliable method to noninvasively detect clarithromycin-resistant H. pylori infection in children. It may be useful before choosing regimens of H. pylori eradication.
authors.affiliation: Department of Pediatrics, Wakayama Medical University, Wakayama, Japan.
authors.forename: Mina
authors.initials: M
authors.lastname: Booka
authors.forename: Masumi
authors.initials: M
authors.lastname: Okuda
authors.forename: Kouichirou
authors.initials: K
authors.lastname: Shin
authors.forename: Eikichi
authors.initials: E
authors.lastname: Miyashiro
authors.forename: Hiroko
authors.initials: H
authors.lastname: Hayashi
authors.forename: Koji
authors.initials: K
authors.lastname: Yamauchi
authors.forename: Yoshitaka
authors.initials: Y
authors.lastname: Tamura
authors.forename: Norishige
authors.initials: N
authors.lastname: Yoshikawa
mesh_terms: D000293:Adolescent; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D000942:Antigens, Bacterial / Q000032:analysis / Q000235:genetics; D002648:Child; D002675:Child, Preschool; D017291:Clarithromycin / Q000494:pharmacology*; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D024881:Drug Resistance, Bacterial; D005243:Feces / Q000382:microbiology*; D016481:Helicobacter Infections / Q000175:diagnosis / Q000382:microbiology; D016480:Helicobacter pylori / Q000187:drug effects* / Q000302:isolation & purification; D006801:Humans; D007223:Infant; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D012680:Sensitivity and Specificity
chemical_list: D000900:Anti-Bacterial Agents; D000942:Antigens, Bacterial; D004269:DNA, Bacterial; D017291:Clarithromycin