P. Deschaght

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

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.

Rapid genotyping of Achromobacter xylosoxidans, Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa and Stenotrophomonas maltophilia isolates using melting curve analysis of RAPD-generated DNA fragments (McRAPD)

Typing of bacteria is important for monitoring newly emerging pathogens and for examining local outbreaks. We evaluated the randomly amplified polymorphic DNA technique in combination with melting curve analysis (McRAPD) of the amplified DNA fragments to genotype isolates from five Gram-negative species, i.e. Achromobacter xylosoxidans, Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa and Stenotrophomonas maltophilia. By determining the melting temperature peaks of the amplified DNA fragments, we were able to distinguish the different genotypes of isolates, as they had been assessed by other genotyping techniques, i.e. agarose gel electrophoresis of RAPD fragments, multilocus sequence typing and/or AFLP™. According to our results, McRAPD may offer the possibility of genotyping a limited number of bacterial isolates, e.g. in case of suspicion of hospital outbreak, via a less costly, more rapid, less laborious and more user-friendly technique than RAPD followed by electrophoresis.

The prevalence of middle ear pathogens in the outer ear canal and the nasopharyngeal cavity of healthy young adults

Culturing middle ear fluid samples from children with chronic otitis media with effusion (OME) using standard techniques results in the isolation of bacterial species in approximately 30-50% of the cases. Haemophilus influenzae, Streptococcus pneumoniae and Moraxella catarrhalis, the classic middle ear pathogens of acute otitis media, are involved but, recently, several studies suggested Alloiococcus otitidis as an additional pathogen. In the present study, we used species-specific PCRs to establish the prevalence, in both the nasopharyngeal cavity and the outer ear, of H. influenzae, M. catarrhalis, S. pneumoniae and A. otitidis. The study group consisted of 70 healthy volunteers (aged 19-22 years). The results indicate a high prevalence (>80%) of A. otitidis in the outer ear in contrast to its absence in the nasopharynx. H. influenzae was found in both the outer ear and the nasopharynx (6% and 14%, respectively), whereas S. pneumoniae and M. catarrhalis were found only in the nasopharynx (9% and 34%, respectively).A. otitidis, described as a fastidious organism, were able to be cultured using an optimized culture protocol, with prolonged incubation, which allowed the isolation of A. otitidis in five of the nine PCR-positive samples out of the total of ten samples tested. Given the absence of the outer ear inhabitant A. otitidis from the nasopharynx, its role in the aetiology of OME remains ambiguous because middle ear infecting organisms are considered to invade the middle ear from the nasopharynx through the Eustachian tube.

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

BackgroundPseudomonas 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.ResultsIn 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.ConclusionBased 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.

Granulomatous meningoencephalitis associated with Sporobolomyces roseus in a dog.

A German Shepherd Dog was evaluated for clinical signs of multifocal, progressive brain disease. Despite supportive care, the dog died shortly after hospital admission. Granulomatous meningoencephalitis with intralesional fungal hyphae was diagnosed postmortem. The fungus was identified as Sporobolomyces roseus by polymerase chain reaction amplification and sequencing of the ITS2 region.