title: Comparison of the sensitivity of culture, PCR and quantitative real-time PCR for the detection of Pseudomonas aeruginosa in sputum of cystic fibrosis patients.
abstract: BACKGROUND
  Pseudomonas aeruginosa is the major pathogen involved in the decline of lung function in cystic fibrosis (CF) patients. Early aggressive antibiotic therapy has been shown to be effective in preventing chronic colonization. Therefore, early detection is important and sensitive detection methods are warranted. In this study, we used a dilution series of P. aeruginosa positive sputa, diluted in a pool of P. aeruginosa negative sputa, all from CF patients--to mimick as closely as possible the sputa sent to routine laboratories--to compare the sensitivity of three culture techniques versus that of two conventional PCR formats and four real-time PCR formats, each targeting the P. aeruginosa oprL gene. In addition, we compared five DNA-extraction protocols.
  
  
  RESULTS
  In our hands, all three culture methods and the bioMérieux easyMAG Nuclisens protocol Generic 2.0.1, preceded by proteinase K pretreatment and followed by any of the 3 real-time PCR formats with probes were most sensitive and able to detect P. aeruginosa up to 50 cfu/ml, i.e. the theoretical minimum of one cell per PCR mixture, when taking into account the volumes used in this study of sample for DNA-extraction, of DNA-elution and of DNA-extract in the PCR mixture.
  
  
  CONCLUSIONS
  In this study, no difference in sensitivity could be found for the detection of P. aeruginosa from sputum between microbiological culture and optimized DNA-extraction and real-time PCR. The results also indicate the importance of the optimization of the DNA-extraction protocol and the PCR format.
authors: Laboratory for Bacteriology Research, Ghent University Hospital, University of Ghent, Ghent, Belgium. deschaght.pieter@ugent.be
authors: Pieter
authors: P
authors: Deschaght
authors: Thierry
authors: T
authors: De Baere
authors: Leen
authors: L
authors: Van Simaey
authors: Sabine
authors: S
authors: Van Daele
authors: Frans
authors: F
authors: De Baets
authors: Daniel
authors: D
authors: De Vos
authors: Jean-Paul
authors: JP
authors: Pirnay
authors: Mario
authors: M
authors: Vaneechoutte
mesh_terms: D001431:Bacteriological Techniques / Q000592:standards; D003550:Cystic Fibrosis / Q000150:complications*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000592:standards; D011552:Pseudomonas Infections / Q000150:complications* / Q000175:diagnosis*; D011550:Pseudomonas aeruginosa / Q000502:physiology; D012680:Sensitivity and Specificity; D013183:Sputum / Q000382:microbiology*