title: Detection of mixed clarithromycin-resistant and -susceptible Helicobacter pylori using nested PCR and direct sequencing of DNA extracted from faeces.
abstract: The major cause of chemotherapy failure in patients with chronic gastritis and peptic ulcers caused by Helicobacter pylori is clarithromycin (CAM) resistance due to a mutation in the 23S rRNA gene. This study describes a non-invasive and accurate method for the detection of mixed CAM-resistant and -susceptible H. pylori by sequencing of the H. pylori 23S rRNA gene. Faeces were crushed with beads and the 23S rRNA gene was amplified using a nested PCR on the extracted DNA. Mutation analysis of this gene using this method showed that 20.4 % of patients carried mixed CAM-susceptible (wild type) and -resistant (A2142G or A2143G mutant) H. pylori. Furthermore, it was found that 66.6 % of patients who had been treated unsuccessfully carried one of these mutations in the 23S rRNA gene (including the mixed type), whilst standard culture detected CAM-resistant isolates in only 22.2 % of patients with unsuccessful treatment. These data suggest that, for successful therapy, the diagnosis method described here would more accurately detect CAM-resistant H. pylori, including mixed infections.
authors.affiliation: Department of Microbiology, School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.
authors.forename: Norihisa
authors.initials: N
authors.lastname: Noguchi
authors.affiliation: Department of Microbiology, School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.
authors.forename: Emiko
authors.initials: E
authors.lastname: Rimbara
authors.affiliation: Department of Microbiology, School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.
authors.forename: Ayami
authors.initials: A
authors.lastname: Kato
authors.affiliation: Third Department of Internal Medicine, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka, Tokyo 181-8611, Japan.
authors.forename: Akifumi
authors.initials: A
authors.lastname: Tanaka
authors.affiliation: Third Department of Internal Medicine, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka, Tokyo 181-8611, Japan.
authors.forename: Kengo
authors.initials: K
authors.lastname: Tokunaga
authors.affiliation: Endoscopy Center, Tokyo Medical University, 6-7-1 Nishishinjuku, Shinjuku, Tokyo 160-0023, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Kawai
authors.affiliation: Third Department of Internal Medicine, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka, Tokyo 181-8611, Japan.
authors.forename: Shin'ichi
authors.initials: S
authors.lastname: Takahashi
authors.affiliation: Department of Microbiology, School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.
authors.forename: Masanori
authors.initials: M
authors.lastname: Sasatsu
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D017291:Clarithromycin / Q000494:pharmacology*; D004269:DNA, Bacterial / Q000235:genetics; D004275:DNA, Ribosomal / Q000235:genetics; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D005243:Feces / Q000382:microbiology*; D005260:Female; D016480:Helicobacter pylori / Q000187:drug effects* / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D008297:Male; D008875:Middle Aged; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D012338:RNA, Ribosomal, 23S / Q000235:genetics; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012338:RNA, Ribosomal, 23S; D017291:Clarithromycin