Comparison of the sensitivity of culture, PCR and quantitative real-time PCR for the detection of Pseudomonas aeruginosain sputum of cystic fibrosis patients

Abstract

BackgroundPseudomonas 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.ResultsIn 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.ConclusionIn 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.

Does bacterial density in cystic fibrosis sputum increase prior to pulmonary exacerbation?

“…Culture-independent analysis of respiratory samples was performed using quantitative (Q)-PCR, which offers many potential advantages over cultivation-based approaches to bacterial enumeration, and has been employed extensively in a wide range of contexts [21]. Whilst a number of key issues must be considered when using these culture-independent techniques, such as the potential for incomplete cell lysis, nucleic acid degradation, and PCR bias or inhibition [35], Q-PCR has been shown to be accurate and reproducible in the analysis of CF airway samples [22,23]. Despite this, molecular quantification of viable bacteria in CF respiratory secretions is challenging due to the persistence of bacterial DNA from dead or lysed cells in airway mucus [24,36].…”

“…Q-PCR technology is well-validated and used widely in the characterisation of bacteria in clinical samples [21], and has been shown to be a sensitive, accurate and reproducible method of bacterial enumeration in samples of CF airway secretions [22,23]. However, where clearance of material from the site of infection is poor, as in the lower CF airways [24], the presence of nonviable cells and extracellular DNA can have a substantial impact on the accuracy of quantification by molecular methods.…”

Clinical, Microbiological and Inflammatory Evidence of the Efficacy of Combination Therapy Including Serratiopeptidase in the Treatment of Periimplantitis

“…In those cases of PI that proved refractory to combined treatment at 6 months, microbiological samples were also processed to evaluate the presence of common bacterial pathogens by molecular methods as described elsewhere (25,26 …”

Development and application of a loop-mediated isothermal amplification method on rapid detection of Pseudomonas aeruginosa strains

“…PCR and real-time PCRbased assays have been employed and developed for the past decades (Schwartz et al 2006;Lodeng et al 2006;Deschaght et al 2009). However, disadvantages for PCR (time consumption for post determination, risk of contamination and low levels detection limit) and real-time PCR (requirement for trained personnel, operating space, expensive equipment and reagents) posed significant obstacles for their broad application.…”