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Journal of Clinical Microbiology, January 2003, p. 397-402, Vol. 41, No. 1
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.1.397-402.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Real-Time PCR Assay for Rapid and Accurate Detection of Point Mutations Conferring Resistance to Clarithromycin in Helicobacter pylori

Monica Oleastro,1,2 Armelle Ménard,1 Adriana Santos,1 Hervé Lamouliatte,3 Lurdes Monteiro,2 Philippe Barthélémy,4 and Francis Mégraud1*

Astra Zeneca France, Rueil Malmaison,4 Laboratoire de Bactériologie-Enfants, Université Victor Segalen Bordeaux 2,1 Gastroentérologie, Hôpital Saint-André, Bordeaux, France,3 Unidade Helicobacter/Campylobacter, Instituto Nacional de Saúde, Lisbon, Portugal2

Received 5 August 2002/ Returned for modification 1 October 2002/ Accepted 27 October 2002

The main cause of failure of Helicobacter pylori eradication therapy is resistance to clarithromycin. The resistance is due to three point mutations in two positions on the 23S rRNA (A2142C, A2142G, and A2143G). Our aim was to develop a rapid and accurate method to detect these mutations directly on biopsy specimens. We developed a real-time PCR that included a simultaneous detection of the amplicons by hybridization of two probes labeled with LC-Red and fluorescein by using the fluorescence resonance energy transfer (FRET) technology and melting curve analysis with the LightCycler thermocycler. The assay was first applied successfully on reference strains, reference plasmids, and H. pylori-negative biopsies. Biopsies from 200 patients having failed a first eradication attempt and for whom the H. pylori strain was available were then tested with the new assay. A result was obtained in 199 cases; a single genotype was detected in 157 cases, two genotypes were detected in 41 cases, and three genotypes were detected in one case. There were, in total, seven discrepancies between the real-time PCR and the phenotypic method of determination of clarithromycin susceptibility, and in an additional four cases the two phenotypic methods were in disagreement. PCR-restriction fragment length polymorphism was applied to a sampling of biopsies, including all of the cases with multiple genotypes and all the cases with discrepant results. Finally, in four cases with discrepant results, the real-time PCR detected the resistant population at a concentration so low that it could not be detected by the phenotypic method, while in three cases other mutations could be involved. This assay had an accuracy at least as satisfactory as that of the phenotypic tests and could be performed within 2 h, allowing it to be used before the administration of therapy in the case of a first H. pylori eradication.

* Corresponding author. Mailing address: Laboratoire de Bactériologie, Hôpital Pellegrin, Place Amélie Raba-Léon, 33076 Bordeaux cedex, France. Phone: 33 (0) 5 56 79 59 10. Fax: 33 (0) 5 56 79 60 18. E-mail: francis.megraud{at}chu-bordeaux.fr.

Journal of Clinical Microbiology, January 2003, p. 397-402, Vol. 41, No. 1
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.1.397-402.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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