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J Med Microbiol 56 (2007), 1174-1180; DOI: 10.1099/jmm.0.47302-0
© 2007 Society for General Microbiology
ISSN 1473-5644

Detection of mixed clarithromycin-resistant and -susceptible Helicobacter pylori using nested PCR and direct sequencing of DNA extracted from faeces

Norihisa Noguchi1, Emiko Rimbara1, Ayami Kato1, Akifumi Tanaka2, Kengo Tokunaga2, Takashi Kawai3, Shin'ichi Takahashi2 and Masanori Sasatsu1

1 Department of Microbiology, School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan

2 Third Department of Internal Medicine, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka, Tokyo 181-8611, Japan

3 Endoscopy Center, Tokyo Medical University, 6-7-1 Nishishinjuku, Shinjuku, Tokyo 160-0023, Japan

Correspondence
Norihisa Noguchi
noguchin{at}ps.toyaku.ac.jp

Received 22 March 2007
Accepted 19 May 2007

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.

Abbreviations: CAM, clarithromycin; HpSA, H. pylori stool antigen.




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