Comparison of the sensitivity of culture, PCR and quantitative real-time PCR for the detection of <it>Pseudomonas aeruginosa </it>in sputum of cystic fibrosis patients

• Main Authors: De Vos Daniel, De Baets Frans, Van daele Sabine, Van Simaey Leen, De Baere Thierry, Deschaght Pieter, Pirnay Jean-Paul, Vaneechoutte Mario
• Format: Article
• Language: English
• Published: BMC 2009-11-01
• Series: BMC Microbiology
• Online Access: http://www.biomedcentral.com/1471-2180/9/244

<p>Abstract</p> <p>Background</p> <p><it>Pseudomonas aeruginosa </it>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 <it>P. aeruginosa </it>positive sputa, diluted in a pool of <it>P. aeruginosa </it>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 <it>P. aeruginosa oprL </it>gene. In addition, we compared five DNA-extraction protocols.</p> <p>Results</p> <p>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 <it>P. aeruginosa </it>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.</p> <p>Conclusion</p> <p>In this study, no difference in sensitivity could be found for the detection of <it>P. aeruginosa </it>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.</p>