Experimental Human Pneumococcal Carriage

1. Volunteer Selection and Screening

1. Healthy volunteers are recruited through poster advertisements and on the University website according to the NHS Research and Ethics Committee guidance.
2. The inclusion criteria for participation are:
   1. Adults aged 18-60 years.
   2. Speaks fluent English and is able to communicate easily by both mobile telephone and text messaging.
3. The exclusion criteria for participation are:
   1. Close contact with at risk individuals (children, immunosuppressed adults, elderly, chronic ill health).
   2. Current smoker or significant smoking history (>10 pack years).
   3. Asthma or other respiratory disease.
   4. Pregnancy.
   5. Allergy to penicillin or amoxicillin.
   6. Current involvement in another clinical trial unless observational or in non-interventional phase.
   7. Unable to give fully informed consent.
4. An initial screening visit, approximately one week before inoculation, includes a focused clinical history and targeted clinical examination.
5. If a previously unrecognized abnormality is found, the volunteer will be excluded from the study and appropriate investigation will be arranged through primary care.
6. During the initial screening visit a full blood count is obtained to ensure that the white cell count is within normal range before the inoculation visit. A nasal wash is performed to exclude natural pneumococcal carriers (see 6.1).
7. Prior to inoculation, volunteers are educated on the risks involved with participation in the study and are provided with an emergency patient leaflet, digital thermometer, emergency telephone numbers and 3 day course of amoxicillin.

2. Preparation of Inoculation Stocks

1. Preparation of the inoculation stocks must be done in a clean environment. The pipettes used should only be used for inoculum preparation. All glass and plastic ware should be sterile. We recommend that inoculation stocks be prepared in a dedicated room, using a dedicated fumehood and incubator.
2. Pneumococcal strains are inoculated onto a blood agar plate. Plates are incubated at 37 °C in 5% CO₂ overnight. The following day all the bacteria is scraped from the blood agar plate and inoculated into a bead stock preservation system. These bead stocks are used for inoculation stock preparation. Test the bead stocks for contamination and colony uniformity before preparing an inoculation stock.
3. To prepare the inoculation stock, inoculate a blood agar plate with two beads from the desired pneumococcal serotype bead stock, streaking to cover the entire plate. Incubate plates at 37 °C in 5% CO₂ overnight. All further incubations will use these same conditions. Also incubate the Vegitone media overnight to ensure sterility.
4. The next morning swab half the plate, taking care not to remove any blood agar, and add to 12 ml of pre-warmed Vegitone broth. Repeat using the other half of the plate. Leave the two 12 ml vials at 37 °C for 2 hr or until a change in turbidity becomes apparent, whichever comes first.
5. Add the 12 ml to 40 ml of pre-warmed Vegitone broth and mix thoroughly. Repeat for the other 12 ml vial. This is time point = 0. Remove 100 μl to read the optical density (OD) at 600 nm. Remove 20 μl for quantification of bacteria by colony forming unit (CFU) count using a variation of the method by Miles and Misra (M&M)⁷. Incubate both vials.
   1. For the M&M, divide a blood agar plate into six sections. Add 180 μl of sterile PBS to six wells of a 96 well plate (U-bottom). Add 20 μl of the broth to the top well and mix. Serially dilute the sample 1:10 six times and discard 20 μl from the sixth well. Place three 10 μl drops from the top well into the first section of the blood agar plate. Repeat for the five other wells. Ensure the plate is dry before it is inverted and incubated.
   2. The next day count the number of visible colonies in each dilution section and record. Using the dilution section with a count between 30 and 300, divide the number of visible colonies by three to get an average.
   3. Multiply the average number of colonies by the dilution factor and then divide by 10 (the amount of the 10 μl drop). This gives CFU/μl.
   4. Multiply this number by 1,000 to get CFU/ml.
6. Perform OD 600 readings and quantification of bacteria by M&M method every hour until early mid-log phase is reached, an OD of 0.25.
7. Once this OD is reached, add sterilized 10% glycerol to one vial and prepare 1 ml aliquots. Store aliquots at -80 °C.
8. For a more concentrated stock, centrifuge the other vial at 3,345 x g for 15 min and remove the supernatant. Resuspend the pellet in 22.5 ml of Vegitone medium and add 10% glycerol. Prepare 1 ml aliquots and store at -80 °C.
9. Leave stocks at -80 °C for at least 48 hr before thawing and quantifying.
10. Quantify the frozen bacterial stock by the M&M method. Test three stock tubes individually to ensure reproducibility. Use the value obtained to dilute the stock to the desired inoculum concentration in saline, as described below.
11. For an inoculation, the quantity of pneumococci in the inoculum is calculated before and after the inoculation. This quantification of bacteria by the M&M method is used to determine the average number of pneumococci in the inoculum which accounts for the time it takes to complete the entire procedure.
    1. Prepare an inoculum, diluting to the desired concentration, and perform quantification by the M&M method for the “pre-inoculation” count.
    2. In our Clinical Research Facility it takes the team 30 min to complete the entire inoculation procedure. Pneumococcal suspensions in saline will usually show a decreased count in this interval. To account for this, we let the diluted inoculum sit at room temperature for 30 min and then perform a “post-inoculation” quantification of the bacteria by the M&M method.
    3. Take the average of the before and after to determine the final inoculated concentration. Adjust the “sitting” time according to your clinical protocol but keep the interval as brief as possible.
12. All new stocks should be sent to a reference laboratory for confirmation of stock purity and identity.

3. Inoculum Preparation

1. Preparation of the inoculum should begin 30 min before the scheduled volunteer inoculation appointment.
2. Take one of the previously made stock tubes out of the -80 °C freezer, thaw it and spin the tube in the microcentrifuge at 17,000 x g for 3 min.
3. Warm blood agar plates in the 37 °C incubator. Prepare a 96 well plate for bacterial quantification by M&M method and prepare dilution tubes according to the desired dose.
4. Remove the broth supernatant from the centrifuged tube and resuspend the pellet in 1 ml of saline. This step removes the broth from the inoculum.
5. Repeat the centrifugation step.
6. Remove the saline supernatant and resuspend the pellet in 1 ml of saline.
7. Perform dilution set for desired inoculum concentration and quantify by M&M method.
8. Take the inoculum to the volunteer appointment and after inoculating the volunteers, repeat the quantification at the lab. Label the tube with the date and inoculum dose and store at -80 °C.
9. To ensure consistency from inoculation to inoculation, it is important to use the same pipettes and dedicated laboratory equipment every time.

4. Inoculation

1. Volunteers should be seated comfortably in a semi-recumbent position.
2. Using a P200 pipette and sterile filter tips draw up 100 μl of the inoculum and insert the tip just inside the nasal cavity.
3. Slowly expel the inoculum onto the nasal mucosa using a circular motion.
4. It is crucial that the pipette tip does not come in contact with the nasal mucosa as a disruption in the integrity of the epithelium could result in the bacteria entering the bloodstream.
5. It is also vital that the inoculum is not placed too far back or it will run down the throat; it should reside inside the nasal cavity.
6. Repeat for the other naris.
7. Have the volunteer remain in the semi-recumbent position for 10 min without sniffing or blowing the nose. This allows time for the bacteria to disperse across the mucosa.

5. Monitoring

1. Volunteers are monitored daily following inoculation. This includes a daily text message sent by the volunteer to investigators before 2 pm.
2. If the text is not received by 2 pm the volunteer will be contacted to ensure his/her well-being. If he/she does not respond, the allocated next of kin will be contacted to ensure the volunteer’s well-being.
3. Volunteers are asked to report on any upper respiratory tract symptoms at every appointment. Clinical staff will ask the volunteer to describe his/her symptoms and will follow up any complaints.
4. The antibiotics given to the volunteers are only to be taken under three circumstances; in the event that they are unwell and are instructed to take them by the research team, if they are carrying pneumococcus at the end of the study, or if they are unwell and unable to contact the research team. The volunteer is encouraged to keep this emergency pack with them at all times during the study and is requested to contact the research team on a daily basis for the next 7 days.
5. Our team is available 24/7 with nurse contact during working hours and two doctors available out of hours. All queries are dealt with by telephone call and/or immediate review. Provisions are available for immediate, direct admission to an Infectious Disease ward, if needed.

6. Nasal Wash (NW) Procedure

1. Nasal washes (NW) are performed at an initial screening visit, as well as 48 hr, 7, and 14 days post- inoculation. The NW method used is taken from Naclerio et al.⁸ Volunteers naturally colonized by S. pneumoniae are excluded from inoculation and follow up.
2. The volunteer is seated comfortably and the head is tilted back 30° from the vertical.
3. Ask the volunteer to take a deep breath in and hold their breath whilst pushing their tongue up and backwards against the roof of the mouth. Whilst in this position, the volunteer is asked to signal that they are ready.
4. A syringe filled with 20 ml saline is inserted into the anterior nasal space and 5 ml of saline is expelled. The volunteer then leans forward immediately and expels the fluid by exhaling rapidly through their nose into a foil bowl.
5. Repeat this procedure 3 more times so that each naris has been washed twice and the full 20 ml has been used.
6. Pool all samples together in a centrifuge tube and send to the laboratory at room temperature for processing.

7. Nasal Wash Processing

1. Centrifuge the samples for 10 min at 3,345 x g.
2. Remove the supernatant and store as 1 ml aliquots in labelled Eppendorf tubes at -80 °C.
3. Add 100 μl of STGG medium⁹ to the pellet and mix thoroughly. Ensure that the total volume in the tube at this point is determined. This dilution will be used in calculating the CFU of a carriage positive NW.
4. Plate a 20 μl drop of the STGG containing the resuspended pellet onto a blood agar plate containing gentamicin and streak the entire plate.
   1. If the NW is post-inoculation, remove 10 μl from the STGG containing the resuspended pellet and use for bacterial quantification by M&M method.
5. Add another 800 μl of STGG to the NW tube and mix thoroughly.
6. Plate 25 μl onto a blood agar plate and 25 μl onto a chocolate agar plate, streaking the entire plate.
7. Divide the remaining amount bacteria suspended in STGG medium into 2 cryovials and store at -80 °C.
8. Incubate the plates at 37 °C overnight in 5% CO₂.
9. Examine the plates the next day for pneumococcus and other potential respiratory pathogens.
10. Pneumococci are identified on plates by colony morphology. Alpha haemolytic colonies with draughtsman morphology are sub-cultured onto blood agar with an optochin disc and incubated overnight.
11. Presumptive pneumococcal colonies that are optochin sensitive are Gram stained to confirm Gram positive diplococci and serotyped using the Statens Serum Institut Pneumotest-Latex kit.