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Optimum Condition for Storage of Sputum from Patients with Bronchiectasis Infected with Haemophilus influenzae

Adam T Hill, Yang Zhang, Katharina Singh Kang, Andrea Clarke, Samantha Donaldson, Manjit Cartlidge, Pallavi Bedi, Catherine J Doherty, Thamarai Schneiders and John RW Govan

Background: The optimum storage condition is not known for the isolation of H. influenzae in bronchiectasis patient sputum samples, the most common pathogen among bronchiectasis patients. The aim of the study was to assess the optimum sputum storage conditions for the isolation of Haemophilus influenzae from microbiological culture in patients with bronchiectasis.

Method and findings: 20 stable bronchiectasis patients were recruited. The first three patients were used to assess reproducibility. 11 patient sputum samples were then collected and then stored for 2, 4, 6, 24 and 48 hours at room temperature and for 24 and 48 hours at 4°C, -20°C or -70°C before processing. The final 6 patient sputum samples were stored in 25% glycerol, 50% skimmed milk and neat at 4°C, -20°C and -70°C for 7 days before processing. Bacteria were identified and median log10 bacterial density (c.f.u./ml-1) and viable rates were calculated. There was good reproducibility with the coefficient of variation varying between 2-9%. The median baseline density of H. influenzae at room temperature was 1.2×108 c.f.u. ml-1. There was no significant difference in the density of H. influenzae cultured from the samples stored at room temperature and 4°C for any of the time points up to 48 h (room temperature: 48 h, 1.0×108 c.f.u. ml-1 and at 4°C: 48 h, 6.5×107 c.f.u. ml-1). Viable rates were the highest in samples left at room temperature for 48 h (81%). After 7 days of storage, only the sample stored at -70°C in glycerol (5.2×107 c.f.u. ml-1) showed no significant difference in the density of H. influenzae compared to processing right after collection (1.2×108 c.f.u. ml-1) and viable rates of 91%.

Conclusion: The optimum conditions for sputum storage and processing for the isolation of H. influenzae from bronchiectasis patients are the storage at room temperature for up to 48 h and storage at -70°C in glycerol for up to 7 days.