title: Evaluation of dual-color fluorescence in situ hybridization with peptide nucleic acid probes for the detection of Mycobacterium tuberculosis and non-tuberculous mycobacteria in clinical specimens.
abstract: BACKGROUND
  Peptide nucleic acid (PNA) probes are artificial DNA analogues with a hydrophobic nature that can penetrate the mycobacterial cell wall. We evaluated a FISH method for simultaneous detection and identification of Mycobacterium tuberculosis (MTB) and non-tuberculous mycobacteria (NTM) in clinical respiratory specimens using differentially labeled PNA probes.
  
  
  METHODS
  PNA probes targeting the mycobacterial 16S ribosomal RNA were synthesized. The cross-reactivity of MTB- and NTM-specific probes was examined with reference strains and 10 other frequently isolated bacterial species. A total of 140 sputum specimens were analyzed, comprising 100 MTB-positive specimens, 21 NTM-positive specimens, and 19 MTB/NTM-negative specimens; all of them were previously confirmed by PCR and culture. The PNA FISH test results were graded by using the United States Centers for Disease Control and Prevention-recommended scale and compared with the results from the fluorochrome acid-fast bacterial stain.
  
  
  RESULTS
  The MTB- and NTM-specific PNA probes showed no cross-reactivity with other tested bacterial species. The test results demonstrated 82.9% agreement with the culture results with diagnostic sensitivity of 80.2% and diagnostic specificity of 100.0% (kappa=0.52, 95% confidence interval: 0.370-0.676).
  
  
  CONCLUSIONS
  Dual-color PNA FISH showed high specificity for detecting and identifying mycobacteria in clinical specimens. However, because of its relatively low sensitivity, this method could be more applicable to culture confirmation. In application to direct specimens, the possibility of false-negative results needs to be considered.
authors.affiliation: Department of Laboratory Medicine, Pusan National University Yangsan Hospital, Yangsan, Korea.
authors.forename: Namhee
authors.initials: N
authors.lastname: Kim
authors.affiliation: Department of Laboratory Medicine, Cheukchu Hospital, Changwon, Korea.
authors.forename: Seung Hee
authors.initials: SH
authors.lastname: Lee
authors.affiliation: Department of Laboratory Medicine, Pusan National University Hospital, Busan, Korea.
authors.forename: Jongyoun
authors.initials: J
authors.lastname: Yi
authors.affiliation: Department of Laboratory Medicine, Pusan National University Yangsan Hospital, Yangsan, Korea. cchl@pusan.ac.kr.|Research Institute for Convergence of Biomedical Science and Technology, Pusan National University Yangsan Hospital, Yangsan, Korea.
authors.forename: Chulhun L
authors.initials: CL
authors.lastname: Chang
mesh_terms: D015342:DNA Probes / Q000378:metabolism; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000302:isolation & purification*; D009170:Nontuberculous Mycobacteria / Q000235:genetics / Q000302:isolation & purification*; D020135:Peptide Nucleic Acids / Q000737:chemistry / Q000378:metabolism*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D013183:Sputum / Q000382:microbiology; D014376:Tuberculosis / Q000175:diagnosis / Q000382:microbiology
chemical_list: D015342:DNA Probes; D020135:Peptide Nucleic Acids; D012336:RNA, Ribosomal, 16S
keywords: Fluorescence in situ hybridization; Mycobacterium tuberculosis; Non-tuberculous mycobacteria; Peptide nucleic acids