title: Validation of a fluorescence in situ hybridization method using peptide nucleic acid probes for detection of Helicobacter pylori clarithromycin resistance in gastric biopsy specimens.
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.
authors.affiliation: IBB-Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, University of Minho, Braga, Portugal.
authors.forename: Laura
authors.initials: L
authors.lastname: Cerqueira
authors.forename: Ricardo M
authors.initials: RM
authors.lastname: Fernandes
authors.forename: Rui M
authors.initials: RM
authors.lastname: Ferreira
authors.forename: Mónica
authors.initials: M
authors.lastname: Oleastro
authors.forename: Fátima
authors.initials: F
authors.lastname: Carneiro
authors.forename: Catarina
authors.initials: C
authors.lastname: Brandão
authors.forename: Pedro
authors.initials: P
authors.lastname: Pimentel-Nunes
authors.forename: Mário
authors.initials: M
authors.lastname: Dinis-Ribeiro
authors.forename: Céu
authors.initials: C
authors.lastname: Figueiredo
authors.forename: Charles W
authors.initials: CW
authors.lastname: Keevil
authors.forename: Maria J
authors.initials: MJ
authors.lastname: Vieira
authors.forename: Nuno F
authors.initials: NF
authors.lastname: Azevedo
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001706:Biopsy; D017291:Clarithromycin / Q000494:pharmacology*; D015331:Cohort Studies; D024881:Drug Resistance, Bacterial; D005753:Gastric Mucosa / Q000382:microbiology; D016481:Helicobacter Infections / Q000382:microbiology*; D016480:Helicobacter pylori / Q000187:drug effects* / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008826:Microbial Sensitivity Tests / Q000379:methods; D015345:Oligonucleotide Probes; D020135:Peptide Nucleic Acids; D011446:Prospective Studies; D012189:Retrospective Studies; D012680:Sensitivity and Specificity
chemical_list: D000900:Anti-Bacterial Agents; D015345:Oligonucleotide Probes; D020135:Peptide Nucleic Acids; D017291:Clarithromycin