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Journal of Clinical Microbiology
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Bacteriology

Validation of a Fluorescence In Situ Hybridization Method Using Peptide Nucleic Acid Probes for Detection of Helicobacter pylori Clarithromycin Resistance in Gastric Biopsy Specimens

Laura Cerqueira, Ricardo M. Fernandes, Rui M. Ferreira, Mónica Oleastro, Fátima Carneiro, Catarina Brandão, Pedro Pimentel-Nunes, Mário Dinis-Ribeiro, Céu Figueiredo, Charles W. Keevil, Maria J. Vieira, Nuno F. Azevedo
Laura Cerqueira
aIBB—Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, University of Minho, Braga, Portugal
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Ricardo M. Fernandes
aIBB—Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, University of Minho, Braga, Portugal
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Rui M. Ferreira
bIPATIMUP—Institute of Molecular Pathology and Immunology of the University of Porto, Porto, Portugal
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Mónica Oleastro
cNational Institute of Health Dr. Ricardo Jorge, Lisbon, Portugal
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Fátima Carneiro
bIPATIMUP—Institute of Molecular Pathology and Immunology of the University of Porto, Porto, Portugal
dFaculty of Medicine of the University of Porto, Porto, Portugal
eCentro Hospitalar São João, Department of Pathology, Porto, Portugal
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Catarina Brandão
dFaculty of Medicine of the University of Porto, Porto, Portugal
fPortuguese Oncology Institute Porto, Department Gastroenterology, Porto, Portugal
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Pedro Pimentel-Nunes
dFaculty of Medicine of the University of Porto, Porto, Portugal
fPortuguese Oncology Institute Porto, Department Gastroenterology, Porto, Portugal
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Mário Dinis-Ribeiro
dFaculty of Medicine of the University of Porto, Porto, Portugal
fPortuguese Oncology Institute Porto, Department Gastroenterology, Porto, Portugal
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Céu Figueiredo
bIPATIMUP—Institute of Molecular Pathology and Immunology of the University of Porto, Porto, Portugal
dFaculty of Medicine of the University of Porto, Porto, Portugal
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Charles W. Keevil
gEnvironmental Healthcare Unit, School of Biological Sciences, University of Southampton, Southampton, United Kingdom
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Maria J. Vieira
aIBB—Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, University of Minho, Braga, Portugal
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Nuno F. Azevedo
aIBB—Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, University of Minho, Braga, Portugal
hLEPAE, Department of Chemical Engineering, Faculty of Engineering, University of Porto, Porto, Portugal
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DOI: 10.1128/JCM.00302-13
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ABSTRACT

Here, we evaluated a previously established peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) method as a new diagnostic test for Helicobacter pylori clarithromycin resistance detection in paraffin-embedded gastric biopsy specimens. Both a retrospective study and a prospective cohort study were conducted to evaluate the specificity and sensitivity of a PNA-FISH method to determine H. pylori clarithromycin resistance. In the retrospective study (n = 30 patients), full agreement between PNA-FISH and PCR-sequencing was observed. Compared to the reference method (culture followed by Etest), the specificity and sensitivity of PNA-FISH were 90.9% (95% confidence interval [CI], 57.1% to 99.5%) and 84.2% (95% CI, 59.5% to 95.8%), respectively. In the prospective cohort (n = 93 patients), 21 cases were positive by culture. For the patients harboring clarithromycin-resistant H. pylori, the method showed sensitivity of 80.0% (95% CI, 29.9% to 98.9%) and specificity of 93.8% (95% CI, 67.7% to 99.7%). These values likely represent underestimations, as some of the discrepant results corresponded to patients infected by more than one strain. PNA-FISH appears to be a simple, quick, and accurate method for detecting H. pylori clarithromycin resistance in paraffin-embedded biopsy specimens. It is also the only one of the methods assessed here that allows direct and specific visualization of this microorganism within the biopsy specimens, a characteristic that allowed the observation that cells of different H. pylori strains can subsist in very close proximity in the stomach.

FOOTNOTES

    • Received 4 February 2013.
    • Returned for modification 5 March 2013.
    • Accepted 7 April 2013.
    • Accepted manuscript posted online 17 April 2013.
  • Supplemental material for this article may be found at http://dx.doi.org/10.1128/JCM.00302-13.

  • Copyright © 2013, American Society for Microbiology. All Rights Reserved.
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Validation of a Fluorescence In Situ Hybridization Method Using Peptide Nucleic Acid Probes for Detection of Helicobacter pylori Clarithromycin Resistance in Gastric Biopsy Specimens
Laura Cerqueira, Ricardo M. Fernandes, Rui M. Ferreira, Mónica Oleastro, Fátima Carneiro, Catarina Brandão, Pedro Pimentel-Nunes, Mário Dinis-Ribeiro, Céu Figueiredo, Charles W. Keevil, Maria J. Vieira, Nuno F. Azevedo
Journal of Clinical Microbiology May 2013, 51 (6) 1887-1893; DOI: 10.1128/JCM.00302-13

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Validation of a Fluorescence In Situ Hybridization Method Using Peptide Nucleic Acid Probes for Detection of Helicobacter pylori Clarithromycin Resistance in Gastric Biopsy Specimens
Laura Cerqueira, Ricardo M. Fernandes, Rui M. Ferreira, Mónica Oleastro, Fátima Carneiro, Catarina Brandão, Pedro Pimentel-Nunes, Mário Dinis-Ribeiro, Céu Figueiredo, Charles W. Keevil, Maria J. Vieira, Nuno F. Azevedo
Journal of Clinical Microbiology May 2013, 51 (6) 1887-1893; DOI: 10.1128/JCM.00302-13
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