Helicobacter pylori specific nested PCR assay for the detection of 23S rRNA mutation associated with clarithromycin resistance.
BACKGROUND
Clarithromycin is one of the most important antibiotics for Helicobacter pylori eradication. However, 5-10% of strains are reported to be resistant. It has been shown that one point mutation in the 23S rRNA gene is associated with resistance to clarithromycin.


OBJECTIVE
To establish a polymerase chain reaction (PCR) system which amplifies a segment of the 23S rRNA gene containing the mutation points with primers specific for H pylori, so that H pylori infection and the mutation associated with clarithromycin resistance can be examined simultaneously.


METHODS
To detect H pylori infection and the mutation simultaneously, primers specific for the H pylori 23S rRNA gene were designed based on sequence conservation among H pylori strains and sequence specificity as compared with other bacteria. DNA from 57 cultured strains and from 39 gastric juice samples was amplified in the seminested 23S rRNA PCR. Clinical applicability was evaluated in 85 patients.


RESULTS
DNA samples from 57 cultured strains were all amplified. The novel assay and the urease A PCR agreed in 37/39 gastric juice samples with no false positives. The assay did not amplify the DNA of bacteria other than H pylori. Eight of 85 samples had the mutation before treatment. In clarithromycin based treatment, eradication was achieved in 2/5 (40%) with the mutation and 29/34 (85%) without the mutation.


CONCLUSIONS
The assay using gastric juice is quick (within 12 hours) and non-invasive (endoscopy not required), enabling rapid initiation of appropriate antibiotic treatment.
Maeda
S
S
Second Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Japan.
Yoshida
H
H
Ogura
K
K
Kanai
F
F
Shiratori
Y
Y
Omata
M
M
D000900:Anti-Bacterial Agents; D001483:Base Sequence; D017291:Clarithromycin; D004352:Drug Resistance, Microbial / Q000235:genetics; D004359:Drug Therapy, Combination / Q000627:therapeutic use; D005750:Gastric Juice / Q000382:microbiology; D016481:Helicobacter Infections / Q000175:diagnosis / Q000188:drug therapy; D016480:Helicobacter pylori / Q000187:drug effects / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D012338:RNA, Ribosomal, 23S / Q000235:genetics*; D012680:Sensitivity and Specificity; D017423:Sequence Analysis, RNA
D000900:Anti-Bacterial Agents; D012338:RNA, Ribosomal, 23S; D017291:Clarithromycin