Using scrapings from the inside lining of the nose to investigate carriage of pneumococcal bacteria

The principle objective is to evaluate if repeated mucosal sampling will obtain sufficient tissue for immunological assessment. Nasal cell yield will be measured using the flow cytometer to count the cell populations from the nasal cell samples taken at baseline, day 2, 7, 9 and 14 post-inoculation.

1. To evaluate the effect of repeated sampling of the nasal mucosa (lining of the nose) on the acquisition of pneumococcal colonisation. Pneumococcal colonisation is measured using microbiological techniques. The nasal wash pellet is plated onto agar plates and incubated for 48 hours using our previous published methods. Our previous studies have shown experimental colonisation rates of 40-60% following inoculation. The results of this study will be compared to previous healthy volunteer studies involving inoculation with 6B pneumococcus.
2. To evaluate if repeated mucosal sampling affects colonisation density and duration compared to previous EHPC studies. Pneumococcal colonies are counted from diluted nasal wash by plating the pellet using a Miles and Mizra technique. This is performed at baseline and day 2, 7, 9 and 14 post inoculation. The duration of colonisation is classed as the final timepoint at which pneumococcal bacteria is identified using classical microbiolgical techniques. The density and duration from this study will be compared to results from previous EHPC studies following inoculation of healthy volunteers to determine if the repeated nasal sampling technique has reduced the acquisition rates.
3. To evaluate participant comfort level following repeated mucosal sampling. Participants were asked to complete a Likert-type questionnaire to rate the level of comfort experienced with each mucosal sampling technique.
4. To evaluate the kinetics of nasal inflammatory response during pneumococcal colonisation. Cytokine levels are measured from the nasosorption sample using the Luminex machine. The levels measured on day 2, 7, 9 and 14 will be compared with the levels measured at baseline.
5. To evaluate if nasal sampling of cells causes a transient bacteraemia – patient safety issue. Paired blood cultures will be taken during each visit where nasal cell samples are taken. The first blood culture will be taken prior to nasal sampling and the 2nd blood culture will be taken 15 minutes post nasal cell sampling. These paired blood cultures will be taken at baseline, day 2, 7, 9 and 14 post inoculation. Aerobic and anaerobic blood cultures will be incubated for 48 hours and reported as per hospital guidelines. Presence of any bacteria in the blood culture post sampling will be classed as a possible transient bacteraemia.
6. To evaluate if pneumococcal killing by alveolar macrophages is increased following nasal colonisation. Bronchial alveolar lavage samples will be used to isolate alveolar macrophages. These cells will be incubated with pneumococcal bacteria overnight. The pneumococcal killing will be measured by counting the recovered pneumococcal bacteria following incubation with the macrophages compared to bacteria that were not incubated with macrophages. This demonstrates the number of bacteria killed by the alveolar macrophages. Healthy volunteers that have been colonised with SPN following inoculation will be compared with negative controls.