Detection of mixed clarithromycin-resistant and -susceptible Helicobacter pylori using nested PCR and direct sequencing of DNA extracted from faeces.
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.
Noguchi
Norihisa
N
Department of Microbiology, School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.
Rimbara
Emiko
E
Department of Microbiology, School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.
Kato
Ayami
A
Department of Microbiology, School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.
Tanaka
Akifumi
A
Third Department of Internal Medicine, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka, Tokyo 181-8611, Japan.
Tokunaga
Kengo
K
Third Department of Internal Medicine, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka, Tokyo 181-8611, Japan.
Kawai
Takashi
T
Endoscopy Center, Tokyo Medical University, 6-7-1 Nishishinjuku, Shinjuku, Tokyo 160-0023, Japan.
Takahashi
Shin'ichi
S
Third Department of Internal Medicine, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka, Tokyo 181-8611, Japan.
Sasatsu
Masanori
M
Department of Microbiology, School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.
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*
D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012338:RNA, Ribosomal, 23S; D017291:Clarithromycin