Assessment of sample handling practices on microbial activity in sputum samples from patients with cystic fibrosis

Aim: The aim of this study was to quantitatively and qualitatively assess theeffect of sample storage on the metabolically active microbial community foundin sputum samples from patients with cystic fibrosis (CF).Methods: Sputum samples were collected and split in two equal aliquots one ofwhich was immersed in RNAlater and refrigerated immediately, the secondstored at room temperature for 24 h and RNAlater was subsequently added.mRNA was extracted, and RT-PCR-DGGE and qPCR analysis of the bacterialand fungal communities was carried out.Results: Significant differences in the bacterial communities between the twoprotocols were observed but there were no significant difference seen in the fungalcommunity analyses. Analysis by qPCR demonstrated that room temperaturestorage gave statistically significant increases in eubacteria and Pseudomonas spp.and a statistically significant decrease in those of Haemophilus influenzae.Conclusions: The analysis of metabolically active microbial communities fromCF sputum using molecular techniques indicated that samples should be storedat 4[1]C upon addition of RNAlater to obtain an accurate depiction of the CFlung microbiota. Also, storing respiratory samples at room temperature maycause an over representation of Pseudomonas aeruginosa and mask the presenceof other clinically significant organisms.