Following a 3-week protein treatment (intravenous, 300 ��g/head),

Following a 3-week protein treatment (intravenous, 300 ��g/head), total RNA was extracted from isolated tu
Patients with cystic fibrosis (CF), an autosomal recessively inherited disease caused by a mutation in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, are particularly susceptible Temsirolimus chemical structure to pulmonary infections with Pseudomonas aeruginosa [1,2]. Colonization of the airways of CF patients with P. aeruginosa results in higher morbidity and mortality because of the faster decline of the lung function, especially from the chronic infection phase onwards [3-5]. Detection of colonization and infection by this pathogen as early as possible enables to postpone the chronic infective stage and eventually to achieve the eradication of P. aeruginosa through early treatment.

Indeed, early aggressive antibiotic therapy is now generally accepted as an efficient means to postpone chronic colonization [6,7]. In most routine laboratories detection of bacterial species in respiratory samples is achieved by culture. However, it has been shown that routine culture of sputa from CF patients yields limited microbiological information since it frequently fails to identify the pathogens, which were shown to be present by means of PCR [8]. Furthermore, the correct detection and identification of P. aeruginosa, although in general not a fastidious organism, is not as straightforward as frequently assumed [9,10].

To circumvent culture associated limitations, several molecular assays for the detection of Pseudomonas species have been described [8,11-19], D?ring and colleagues [20] correctly remarked that, because of the influence of sample pretreatment, DNA-extraction protocol and the PCR format, there is a need for validation of the PCR techniques before these can be used in a routine laboratory. However, to our knowledge, no study systematically compared the sensitivity of different culture, DNA-extraction, PCR and real-time PCR methods for the detection of P. aeruginosa from CF sputum, by using a CF patient sputum based dilution series of P. aeruginosa. Here, we compared the sensitivity of three culture media, five DNA-extraction protocols, two conventional PCR formats and four real-time PCR formats for the detection of P. aeruginosa, using a dilution series of P. aeruginosa positive sputa in a pool of P. aeruginosa negative sputa.

Results In this study, we compared the sensitivity of different culture and PCR methods. To that purpose, we prepared a P. aeruginosa dilution series in CF sputum by diluting P. aeruginosa positive CF patient sputa in a pool of P. aeruginosa negative CF patient sputa. This was done instead of diluting cultured P. aeruginosa cells in saline or diluting P. aeruginosa positive sputum in saline or spiking sputa with P. aeruginosa cells, to mimick as closely as possible the sputum samples sent Carfilzomib to routine laboratories.

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