Effect of preparation method and storage period on the stability of saliva DNA.

Saliva is an attractive source for oral microbial detection and quantification since sampling is non-invasive and rapid. OBJECTIVES: To determine whether different saliva preparation methods or preservation time periods affect DNA stability. METHODS: Saliva samples from 4 healthy adult volunteers were processed to obtain 3 different preparations: whole saliva, and after centrifugation pellet and supernatant. Purified DNA (MasterPure™) from each sample was divided into 4 aliquots, one for immediate analysis and 3 (stored at -80°C) for later analyses after 1 week and 2 and 6 months. DNA concentrations and qPCR based quantities of Porphyromonas gingivalis, Prevotella intermedia, Parvimonas micra, Fusobacterium nucleatum, Filifactor alocis and Streptococcus mutans were determined. RESULTS: DNA concentration did not decrease (P>0.05) during the 6-month period in any sample. Mean (SE) DNA concentrations (ng/µl) in whole saliva were 152.2 (51.2) and 147.8 (50) at day 0 and 6 months, respectively. Similarly, the values for pellet were 134.9 (42.5) and 133.6 (42.9), and for supernatant, 11 (1.9) and 8.9 (2.3), the difference being significant (P<0.001) between supernatant and whole saliva or pellet. The quantities of most bacterial species found at day 0 remained stable over the 6-month period in all saliva preparations. In supernatant, species quantities were lower (P<0.05) than in whole saliva or pellet. CONCLUSIONS: DNA concentrations were comparable between whole saliva and pellet, suggesting that either of them can be used for DNA-based analyses. Our results also demonstrated that DNA extracted from saliva can be preserved at -80°C for at least 6 months without decrease in DNA concentration.

Commencement of warfarin therapy in children following the Fontan procedure.

INTRODUCTION: Following the Fontan procedure, children require lifelong anticoagulant therapy to minimise the risk of thrombosis. Factors influencing the management of warfarin therapy in children post-Fontan procedure are poorly understood. This study investigated factors affecting warfarin therapy stability and time to reach target therapeutic range, in children post-Fontan procedure. MATERIALS AND METHODS: Medical histories of children who had undergone Fontan procedures between 1st June 2008 and 1st June 2010 were retrospectively audited. Variables of interest included dietary intake, activity level, gastro-intestinal function, presence of intercostal catheters and antibiotic therapy requirements. Time of warfarin commencement, attainment of a therapeutic International Normalized Ratio (INR) and dosing of warfarin were of particular interest. RESULTS: Decreased warfarin dosage, compared to the clinical practice guidelines, was seen in all children. Antibiotic requirement correlated with an increased hospital length of stay in post-Fontan patients. Requirement for maintenance fluids and immobility lead to a further decrease in the warfarin dose received. CONCLUSIONS: This study presents previously unknown information about the trajectory of warfarin therapy in children post-Fontan procedure. Results contribute to our understanding of the factors that impact warfarin stability in this population. This evidence together with continued research may aid to improve the clinical management of children post-Fontan procedure by reducing the time required to attain target INRs and, potentially length of hospital stay.