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Journal of Clinical Microbiology, October 2004, p. 4512-4518, Vol. 42, No. 10
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.10.4512-4518.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Novel Real-Time PCR Assay for Detection of Helicobacter pylori Infection and Simultaneous Clarithromycin Susceptibility Testing of Stool and Biopsy Specimens

Claudia Schabereiter-Gurtner,1 Alexander M. Hirschl,1 Brigitte Dragosics,2 Peter Hufnagl,3 Sonja Puz,1 Zsuzsanna Kovách,1 Manfred Rotter,1 and Athanasios Makristathis1*

Department of Clinical Microbiology, Institute of Hygiene and Medical Microbiology, Medical University of Vienna,1 The Ambulatory Care Centre South, Regional Public Medical Insurance Agency,2 Roche Diagnostics, Vienna, Austria3

Received 24 February 2004/ Returned for modification 14 April 2004/ Accepted 13 June 2004

A biprobe real-time PCR protocol, followed by hybridization melting point analysis, to detect point mutations in the 23S rRNA gene of Helicobacter pylori associated with clarithromycin resistance was established and evaluated in a clinical study. Of 92 patients who underwent endoscopy, 45 were found to be H. pylori infected and invariably were also culture positive. Of the 45 isolates, 11 were shown to be resistant to clarithromycin by E-test. With respect to the detection of H. pylori infection, PCR showed sensitivities of 100% in biopsies and 98% in stool specimens and a specificity of 98% in both biopsy and stool samples. All clarithromycin-sensitive cases were identified as such by PCR in both biopsy and stool samples. Of the cases with a resistant strain, eight were identified as such in stool DNA and nine were identified in biopsy DNA. Failure of PCR to detect the resistant genotype in the biopsy DNA, stool DNA, or both (one case) was associated with mixed populations. In these cases, patients had not been treated for H. pylori infection before, and the sensitive population showed to be present in considerably higher numbers than the resistant population. In five of six cases in which infection with a resistant genotype only was identified by PCR, the patients had received clarithromycin-based eradication therapy in the past. Thus, the assay presented provides a highly accurate noninvasive method to detect H. pylori infection in stool and at the same time allows for culture-independent clarithromycin susceptibility testing.

* Corresponding author. Mailing address: Department of Clinical Microbiology, University Hospital of Vienna, Währinger Gürtel 18-20, 1090 Vienna, Austria. Phone: 43 (1) 40400-5157. Fax: 43 (1) 40400-5162. E-mail: athanasios.makristathis{at}meduniwien.ac.at.

Journal of Clinical Microbiology, October 2004, p. 4512-4518, Vol. 42, No. 10
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.10.4512-4518.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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