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Journal of Clinical Microbiology, January 2000, p. 210-214, Vol. 38, No. 1
0095-1137/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Detection of Clarithromycin-Resistant Helicobacter pylori Strains by a Preferential Homoduplex Formation Assay

Shin Maeda,1,* Haruhiko Yoshida,1 Hironari Matsunaga,2 Keiji Ogura,1 Osamu Kawamata,3 Yasushi Shiratori,1 and Masao Omata1

Department of Gastroenterology, University of Tokyo,1 and Center for Molecular Biology and Cytogenetics, SRL Inc.,3 Tokyo, and Institute for Biotechnology Research, Wakunaga Pharmaceutical Co. Ltd., Hiroshima,2 Japan

Received 22 July 1999/Returned for modification 23 September 1999/Accepted 23 October 1999

It has been shown that resistance to clarithromycin, a major cause of failure in Helicobacter pylori eradication therapy, is associated with point mutations in the 23S rRNA gene. We sought to apply the preferential homoduplex formation assay (PHFA), a novel technique for the efficient detection of point mutations, to detection of the mutations. PHFA was performed on streptavidin-coated microtiter plates with biotin- and dinitrophenyl-labeled amplicons to detect the wild-type gene or each mutant gene. DNA samples were extracted from gastric juice specimens of 412 patients with H. pylori infection and were applied to the assay. The detection threshold of PHFA was as few as 10 gene copies. The sensitivity of PHFA for the detection of H. pylori infection was higher than those of culture and the rapid urease test. A total of 337 (81.8%) samples had the wild-type gene, 38 (9.2%) had the A2144G mutation, and 37 (9.0%) contained both the wild type and a mutation (A2144G in 30 samples, A2143G in 5 samples, and A2143G plus A2144G in 2 samples). About half the strains isolated from patients with mixed infection were susceptible by the agar dilution method (MIC, <0.1 mg/liter). Therefore, PHFA can detect clarithromycin-resistant H. pylori strains, even in patients with mixed infections with the wild type, that are not detectable by the agar dilution method.

* Corresponding author. Mailing address: Department of Gastroenterology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-8655, Japan. Phone: 81-3-3815-5411, ext. 3070. Fax: 81-3-3814-0021. E-mail: MAEDA-2IM{at}h.u-tokyo.ac.jp.

Journal of Clinical Microbiology, January 2000, p. 210-214, Vol. 38, No. 1
0095-1137/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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