Methodological research of assessing lower airways bacteria in patients with Bronchiectasis
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Aims: The first part of my MScR was to identify the optimum method for identifying bacteria in the lower airways of bronchiectasis patients. The second part was to investigate processing of bronchiectasis patients chronically infected with Haemophilus influenzae. Methods: Part one: 29 patients used four kinds of techniques to sample the lower airways: cough plates, exhaled breath condensate (EBC), induced and spontaneously produced sputum. Bacteria were identified and median sputum bacterial density (cfu./ml-1) was calculated. Part two: 17 patients were recruited. 12 of their spontaneously produced sputum samples were processed as follows: 1 h, 2 h, 4 h, 6 h, 24 and 48 h at room temperature and after 24 and 48 h at 4 °C, -20 °C and -70 °C. The other 6 samples were processed at 1 h as the baseline and stored with glycerol, skimmed milk and in neat in 4°C, -20°C and -70°C for a week before processing. Bacteria were identified and median sputum bacterial density (cfu./ml-1) was calculated. Results: Part one: Bacterial pathogens were isolated in 0% of EBC, 21% of cough plates, 72% of induced sputum, 69% of spontaneous sputum. Part two: From sample being produced up to 48h before processing, there was no significant difference in the density of H.influenzae cultured from the samples stored at room temperature and 4°C for up to 48h when compared to samples processed after 1h. However, a significant reduction was observed for 24h and 48h at -20°C and -70°C. After one week storage before processing, only the sample stored at -70°C with glycerol showed no significant difference in the density of H. influenzae. Other storage conditions led to a reduction in bacterial load. Conclusions: The first study shows that induced sputum and spontaneously produced sputum are all reliable methods to measure bacteria in the lower airways. The second study shows that the ideal sputum storage method for up to 48h is room temperature, and for up to one week is stored with glycerol at -70°C.