JCM Accepts, published online ahead of print on 11 November 2009

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J. Clin. Microbiol. doi:10.1128/JCM.01324-09
Copyright (c) 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Determining CF lung microbiology: a comparison of spontaneous and serially induced sputum samples using T-RFLP profiling

Geraint B. Rogers^*, Stuart Skelton, David J. Serisier, Christopher J. van der Gast, and Kenneth D. Bruce

King's College London, Molecular Microbiology Research Laboratory, Pharmaceutical Science Division, 150 Stamford Street, Franklin-Wilkins Building, London, SE1 9NH, UK; Department of Respiratory Medicine, Mater Adult Hospital, South Brisbane, QLD 4101, Australia; NERC Centre for Ecology and Hydrology, Mansfield Road, Oxford, OX1 3SR, UK

^* To whom correspondence should be addressed. Email: geraint.rogers{at}kcl.ac.uk.

Sampling the lower airways of the adult cystic fibrosis lung has received insufficient detailed consideration, with the importance of sampling strategies on bacteriological outcome not known. Although Spontaneously Expectorated Sputum (SES) samples are often used for diagnostic bacteriological analysis, Induced Sputum (IS) methods have advantages. This study asked whether significant differences in bacterial content were detected when using a culture-independent, molecular profiling technique to analyse SES or IS samples. Moreover, this work asked what trends relating to bacterial species distributions and reproducibility were found in sequentially induced sputum samples, and what implications this has for pathogen detection. T-RFLP analysis was performed on a SES sample and 4 subsequent IS samples taken at 5 minute intervals from 10 clinically stable, adult CF patients. This was repeated over 3 sampling days, with variability between samples, induction periods and sampling days determined. A diverse range of bacterial species, including potentially novel pathogens, were found. No significant difference in bacterial content was observed for either SES or serial IS samples. On average, the analysis of a single sample from any time point resolved 58% of total bacterial diversity achieved by analysis of an SES sample and 4 subsequent IS samples. The reliance on analysis of a single respiratory sample was not sufficient for the detection of recognised CF pathogens in all instances. Close correlation between T-RFLP and microbiological data in the detection of key species indicates the importance of these findings in routine diagnostics for the detection of recognised and novel CF pathogens.