S80 A Clinical Evaluation of Quantitative, Culture-Independent Methods For the Identification of Bacteria in Cystic Fibrosis Sputum and Broncho-Alveolar Lavage Fluids

Abstract

Introduction and objectives: The detection of bacteria contributing to respiratory exacerbations in cystic fibrosis patients is routinely performed using standardised microbiological culture techniques. These methods often fail to identify a significant pathogen in symptomatic patients and have been found to underestimate the burden of fastidious organisms such as Streptococcus pneumoniae in CF sputum. There is also increasing evidence linking respiratory exacerbations to infection with organisms that are not identified by routine microbiology. The primary objective of this cross-sectional study was to test the agreement between standard microbiology and molecular (culture-independent) techniques in detecting five common pathogens in sputum of children with CF. Methods Culture-independent microbiology was performed on samples obtained from children experiencing respiratory exacerbations over a 12 month period from April 2010 to March 2011. Sputum samples were either produced spontaneously or obtained either by sputum induction with hyper tonic saline or bronchoalveolar lavage. Aliquots of sputum we refrozen either neat or mixed 1:1 with 0.1% dithiothreiotol, prior to batch extraction of total nucleic acid. Total bacterial load was determined using abroad-range 16S rDNA quantitative real-time PCR. Specific real-time qPCR assays were used to quantify the numbers of Staphylococcus aureus, Haemophilus influenzae, S pneumoniae, Pseudomonas aeruginosa and Moraxella catarrhalis. Routine microbiology was performed by adhering to the laboratory standards published by the UK CF Trust. Results Fifty five samples (44 sputum and 11 bronchoalveolar lavage fluid) from 33 children were available for molecular analysis. In 40, the total bacterial load was calculated to be 10 7 or greater per ml of fluid. The qPCRs detected clinically relevant pathogens at significant levels (>1% of total) more frequently than standard microbiology. Conclusion We found that standard microbiology alone revealed fewer of the clinically relevant respiratory pathogens studied compared to molecular techniques. PCR based analysis also offers the potential to identify pathogens more rapidly and to detect organisms that are difficult to detect by routine culture. This may have an impact on the choice of antibiotic for the treatment of an exacerbation. Compared to standard microbiology, however, these assays are expensive and not readily available for routine diagnostics.