title: PNA-FISH as a new diagnostic method for the determination of clarithromycin resistance of Helicobacter pylori.
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.
authors.affiliation: IBB - Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, Universidade do Minho, Campus de Gualtar 4710-057, 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: Fátima
authors.initials: F
authors.lastname: Carneiro
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: Nuno F
authors.initials: NF
authors.lastname: Azevedo
authors.forename: Maria J
authors.initials: MJ
authors.lastname: Vieira
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D017291:Clarithromycin / Q000494:pharmacology*; D024881:Drug Resistance, Bacterial; D005838:Genotype; D016480:Helicobacter pylori / Q000187:drug effects* / Q000235:genetics*; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008826:Microbial Sensitivity Tests / Q000379:methods; D058889:Molecular Typing; D015345:Oligonucleotide Probes / Q000235:genetics; D020135:Peptide Nucleic Acids; D012680:Sensitivity and Specificity
chemical_list: D000900:Anti-Bacterial Agents; D015345:Oligonucleotide Probes; D020135:Peptide Nucleic Acids; D017291:Clarithromycin