Comparison of culture and qPCR for the detection of Pseudomonas aeruginosa in not chronically infected cystic fibrosis patients

BMC Microbiology Comparison of culture and qPCR for the detection of Pseudomonas aeruginosa in not chronically infected cystic fibrosis patients Pieter Deschaght 0 Petra Schelstraete 2 Guido Lopes dos Santos Santiago 0 Leen Van Simaey 0 Filomeen Haerynck 2 Sabine Van daele 2 Elke De Wachter 1 Anne Malfroot 1 Patrick Lebecque 6 Christiane Knoop 5 Georges Casimir 5 Hedwige Boboli 4 Frdric Pierart 4 Kristine Desager 3 Mario Vaneechoutte 0 Frans De Baets 2 0 Laboratory for Bacteriology Research (LBR), Ghent University Hospital, Ghent University , Ghent , Belgium 1 Cystic Fibrosis Centre, Universitair Ziekenhuis Brussel , Brussels , Belgium 2 Cystic Fibrosis Centre Ghent, Ghent University Hospital , Ghent , Belgium 3 Cystic Fibrosis Centre Antwerp, University Hospital Antwerp , Antwerp , Belgium 4 Cystic Fibrosis Centre Liege, CHR La Citadelle and CHC Esperance , Liege , Belgium 5 Cystic Fibrosis Centre ULB, Hopital Universitaire des Enfants Reine Fabiola, Erasme University Hospital , Brussels , Belgium 6 Cystic Fibrosis Centre, Cliniques St Luc, University of Louvain , Brussels , Belgium Background: Pseudomonas aeruginosa is the major respiratory pathogen causing severe lung infections among CF patients, leading to high morbidity and mortality. Once infection is established, early antibiotic treatment is able to postpone the transition to chronic lung infection. In order to optimize the early detection, we compared the sensitivity of microbiological culture and quantitative PCR (qPCR) for the detection of P. aeruginosa in respiratory samples of not chronically infected CF patients. Results: In this national study, we followed CF patients during periods between 1 to 15 months. For a total of 852 samples, 729 (86%) remained P. aeruginosa negative by both culture and qPCR, whereas 89 samples (10%) were positive by both culture and qPCR. Twenty-six samples were negative by culture but positive by qPCR, and 10 samples were positive by culture but remained negative by qPCR. Five of the 26 patients with a culture negative, qPCR positive sample became later P. aeruginosa positive both by culture and qPCR. Conclusion: Based on the results of this study, it can be concluded that qPCR may have a predictive value for impending P. aeruginosa infection for only a limited number of patients. - Background Cystic fibrosis (CF) is one of the most common genetic disorders, caused by mutations in the CFTR gene, coding for the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein [1]. Mutations in this gene lead to inactivity of the CFTR protein and/or reduced expression of the protein at the cytoplasmic membrane [2]. Improper functioning of the CFTR results in the production of viscous mucus and in a defective innate immunity [2,3]. The reduced functionality of the mucociliary system and the ongoing inflammation result in an increased sensitivity of the CF airways to infection by bacterial pathogens, of which Pseudomonas aeruginosa and Staphylococcus aureus are the most important. Chronic lung infection with P. aeruginosa is a major cause of morbidity and mortality among the CF patients [4]. It is now well-established that early aggressive antibiotic treatment of new infection with P. aeruginosa is successful in postponing chronic infection. Hence, it is important to detect new infection with P. aeruginosa as early as possible so that eradication treatment can be started as soon as possible [5-7]. Currently, routine detection and identification of P. aeruginosa in respiratory samples is done by conventional methods such as culture and biochemical characteristics. Misidentification can occur due to the variable phenotypic characteristics of this species [8]. Moreover, the sensitivity of culture might be limited, especially when compared to DNA amplification based techniques. Thus far, however, only one group has compared both approaches in a long term study for early detection of P. aeruginosa from CF patients [9]. In this national study, we followed CF patients during periods between 1 to 15 months and we compared the sensitivity of conventional culture techniques with qPCR for the detection of P. aeruginosa in the respiratory samples from CF patients, not chronically infected by P. aeruginosa. which was also incubated for at least 24 h at 37C before examination. After a maximum of 5 days incubation, lactose negative colonies on MacConkey Agar were picked, subcultured onto a 5% Sheep Blood Agar plate (Becton Dickinson) and identified using tDNA-PCR [12]. Methods Patients and sampling From January 2008 until May 2009, sputum, nasopharyngeal or throat swab samples were routinely collected from 397 CF patients attending all but one of Belgian CF-centres, i.e. Ghent University Hospital (UZG, Ghent), Universitair Ziekenhuis Brussel (UZB, Brussels), St Luc University Hospital (UCL, Brussels), Queen Fabiola Childrens University Hospital and Erasme University Hospital (ULB, Brussels), Antwerp University Hospital (UZA, Antwerp), CF Center Liege (CHC - CHR, Liege). Patients were seen every three months and sputum or nasopharyngeal aspirate/throat swab samples were cultured at every visit. Nasopharyngeal aspirates/throat swab samples were collected in case the patients could not expectorate. All 397 included patients, (median age: 14 years, range: 1-53 years), were considered as P. aeruginosa free and not chronically infected according to the criteria used by the different Belgian CF centers, i.e., the European Consensus criteria [10] or those defined by Lee et al. [11]. For the 252 patients with at least two respiratory samples (median: 3 samples, range: 2-11 samples), the median follow-up time was 6 months (range: 115 months). Patients with a P. aeruginosa positive culture were treated according to the standard antibacterial treatment protocols of each center, patients with only a PCR positive result were not treated. Sample processing After arrival at the Laboratory Bacteriology Research (LBR), sputum and nasopharyngeal samples were liquefied with Sputasol (Oxoid Ltd., Basingstoke, UK) (1:1, vol/vol, 1 h incubation at 37C). Throat swabs (ESwab, Copan, Brescia, Italy) were vortexed in the liquid transport medium present in the Eswab tube. For microbiological culture, samples were immediately processed after arrival. For qPCR, at least 200 l of each sample was stored at -80C prior to DNA-extraction. Culture and identification of the bacteria Fifty l of the samples were inoculated onto MacConkey Agar plates (Becton Dickinson, Erembodegem, Belgium) and 100 l into 4 ml Cetrimide Broth (Fluka Biochemika, Buchs, Switzerland) and incubated for at least 24 h at 37C at ambient atmosphere before examination. Cetrimide Broth was subcultured by inoculating 50 l onto a Sheep Blood Agar plate (Becton Dickinson), DNA-extraction Before DNA-extraction, respiratory samples were preincubated with proteinase K, i.e. incubation of 200 l of each sample during 1 h at 55C in 200 l proteinase K buffer ( (...truncated)