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Detection of mutations associated with resistance to tetracycline and clarithromycin in Helicobacter pylori using the Pyrosequencer

Abstract number: 902_p738

Lawson A.

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Objectives:

Clarithromycin and tetracycline are key components of H. pylori eradication therapy. Resistance to clarithromycin occurs due to single nucleotide mutations in 16S rDNA and an assay to detect these was amongst the first to be developed for the Pyrosequencer. Recently it has been shown that resistance and reduced susceptibility to tetracycline occur due to single, double or triple mutations in 23S rDNA. The aim of this study was to develop a single multiplex assay using the Pyrosequencer to determine susceptibility to clarithromycin and tetracycline from H. pylori isolates and direct from gastric biopsy samples.

Methods:

Pyrosequencer assays to detect mutations conferring tetracycline and clarithromycin resistance were designed to work singly and in multiplex. The assays were evaluated using 20 isolates with fully characterised 16S and 23S rDNA sequences. Subsequently, DNA extracts from 30 clinical isolates and 20 H. pylori-positive human gastric biopsies – all of unknown antibiotic susceptibility – were examined and the results compared with those achieved by conventional culture-based techniques, namely antibiotic disc diffusion and Etest.

Results:

The Pyrosquencer multiplex assay correctly determined the 16S and 23S rDNA sequences of the 20 characterised control isolates. When applied to DNA extracted from clinical isolates and gastric biopsy samples, the Pyrosequencer assay was in agreement with the clarithromycin and tetracycline susceptibilities determined by culture-based analysis.

Conclusion:

The Pyrosequencer assay allowed rapid determination of clarithromycin and tetracycline susceptibility from both H. pylori isolates and gastric biopsy samples. The sequence data obtained for the mutations occurring in each strain may provide useful epidemiological information and guide patient management.

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Session Details

Date: 01/08/2007
Time: 00:00-00:00
Session name: XXIst ISTH Congress
Subject:
Location: Oxford, UK
Presentation type:
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