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Open Access Methodology article

PNA-FISH as a new diagnostic method for the determination of clarithromycin resistance of Helicobacter pylori

Laura Cerqueira1, Ricardo M Fernandes1, Rui M Ferreira23, Fátima Carneiro234, Mário Dinis-Ribeiro35, Céu Figueiredo23, Charles W Keevil6, Nuno F Azevedo17 and Maria J Vieira1*

Author Affiliations

1 IBB - Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, Universidade do Minho, Campus de Gualtar 4710-057, Braga, Portugal

2 IPATIMUP - Institute of Molecular Pathology and Immunology of the University of Porto, Porto, Portugal

3 Medical Faculty of the University of Porto, Porto, Portugal

4 Hospital S. João, Department of Pathology, Porto, Portugal

5 Portuguese Oncology Institute Porto, Department Gastroenterology, Porto, Portugal

6 Environmental Healthcare Unit, School of Biological Sciences, University of Southampton, Southampton, UK

7 LEPAE, Department of Chemical Engineering, Faculty of Engineering, University of Porto, Porto, Portugal

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BMC Microbiology 2011, 11:101  doi:10.1186/1471-2180-11-101

Published: 14 May 2011

Abstract

Background

Triple therapy is the gold standard treatment for Helicobacter pylori eradication from the human stomach, but increased resistance to clarithromycin became the main factor of treatment failure. Until now, fastidious culturing methods are generally the method of choice to assess resistance status. In this study, a new genotypic method to detect clarithromycin resistance in clinical samples, based on fluorescent in situ hybridization (FISH) using a set of peptide nucleic acid probes (PNA), is proposed.

Results

The set of probes targeting the point mutations responsible for clarithromycin resistance was applied to H. pylori suspensions and showed 100% sensitivity and specificity (95% CI, 79.9-100 and 95% CI, 71.6-100 respectively). This method can also be amenable for application to gastric biopsy samples, as resistance to clarithromycin was also detected when histological slides were tested.

Conclusions

The optimized PNA-FISH based diagnostic method to detect H. pylori clarithromycin resistance shown to be a very sensitive and specific method for the detection of clarithromycin resistance in the H. pylori smears and also proved to be a reliable method for the diagnosis of this pathogen in clinical samples and an alternative to existing plating methods.