Table 3.

Data collection methods

Determination of colonisation	Colonisation will be defined as result of nasal washes taken at 2, 7, 13, 16, 21 and 28 days post inoculation. Nasal washes will be performed using the Naclerio method,14 which is a validated technique to collect nasal bacterial specimens. Nasal washes will be plated onto culture media. Colonies will be confirmed as SPN3 using classical microbiological techniques. Results from the cultured nasal wash will also be confirmed using PCR-based methods.
Molecular methods of determination of colonisation	DNA will be extracted from bacterial pellet post nasal wash sample centrifugation. SPN3 detection will be done by multiplex qPCR. This technique will enable us to detect individuals who are potential carriers with very low bacterial density. This multiplex qPCR is well validated in our laboratory.16
Viral detection and quantification	Viral multiplex qPCR for detection and quantification will be performed on DNA and RNA of stored throat swab and/or nasal wash to detect all common respiratory viruses.
Mucosal and systemic immune responses	Serotype-specific responses and their association with both acquisition and clearance of colonisation (density and duration) will be measured. We will compare antibody levels and function between those colonised and those protected against colonisation. Levels of immunoglobulin to the capsular polysaccharides SPN3 in serum and nasal washes before and after inoculation will be determined. Levels of SPN3-specific memory B cells will be assessed using PBMCs collected preinoculation and post inoculation. Flow cytometry will be used to examine the induction of antigen-specific cellular responses in blood including B cell and T cells. Mucosal cellular responses will also be measured by flow cytometry on nasal cell samples. Additionally, the mucosal inflammatory response associated with inoculation will be evaluated using 30-plex Luminex method to detect cytokines and chemokines in nasal filters

PBMCs, peripheral blood mononuclear cells; SPN3, Streptococcus pneumoniae serotype 3.