The aim of this procedure is to establish experimental human pneumococcal carriage. This is accomplished by first diluting and quantifying the bacteria in the inoculum. In the second step, the volunteer is intranasally inoculated with streptococcus pneumoniae.
Next, the volunteer has a nasal wash on days two, seven, and 14 after the inoculation. Finally, classical microbiology is used to determine if the volunteer is carrying the inoculated occus. Ultimately, experimental human pneumococcal carriage can be obtained using this safe and reproducible method.
The main advantage of this technique over existing murine carriage models is that this technique offers a model of carriage in the natural host of the pneumococcus the human. Further, this method can help answer key questions in the pneumococcal field, such as what are the immunological correlates of protection? What is the immunizing effect of carriage, and what is the effect of the host pressure on the pathogen in the nasopharyngeal niche?
The applications of this technique include its use as a method to down select novel pneumococcal vaccines. We plan to use the prevention of carriage as a surrogate of vaccine-induced immunity. Demonstrating the clinical technique will be Sister Angie Wright and charge nurse David Shaw.
30 minutes before the scheduled volunteer inoculation appointment tho an inoculum stock tube taken from the minus 80 degrees Celsius freezer. Then spin down the tho inoculum in a micro centrifuge for three minutes at 17, 000 times. G, while the tube is spinning, warm some blood agar plates in a 37 degrees Celsius incubator.
Then remove the broth sate from the centrifuge inoculum, and wash the pellet in one milliliter of saline. Repeat the centrifugation and wash step. Perform a dilution set for the desired inoculum concentration, and then quantify the amount of bacteria in the inoculum by a miles and misra like method.
The grown out colonies will become visible the following day. Take the inoculum to the volunteer who should be seated comfortably in a semi recumbent position. Then using a P 200 perpe and sterile filter tips.
Draw 100 microliters of the inoculum into the perpe tip, and insert the tip just inside the nasal cavity. Slowly expel the inoculum onto the nasal mucosa using a circular motion. Do not place the pipette tip too far back, or the inoculum will run down the throat.
Repeat the inoculation for the other naris to avoid bacteria entering the bloodstream. Be careful not to disrupt the mucosa with the pipette. Then have the volunteer remain in the semi recumbent position for 10 minutes to allow the bacteria to disperse across the mucosa.
After administering the inoculum to the volunteers, repeat the bacterial quantification at the lab. Then label the tube with the date and inoculum dose, and store the inoculum at minus 80 degrees Celsius. Before starting the nasal wash, have the volunteer sit comfortably with the head tilted back 30 degrees from the vertical.
Then have the volunteer take and hold a deep breath while pushing the tongue up and backwards against the roof of the mouth. Have the volunteer signal when this position is achieved. Now wash the nasal cavity four times with 20 milliliters of saline, discharging five milliliters of the saline at a time into the anterior nasal space.
Have the volunteer immediately lean forward and exhale rapidly through the nose into a foil bowl to expel the fluid After each five milliliter saline flush. After washing the nasal cavity with the four 20 milliliters, pull all the collected samples together in a centrifuge tube. Now centrifuge the volunteer nasal wash samples for 10 minutes at 3, 345 times G.Remove the supinate and store it as one milliliter aliquots in labeled einor tubes at minus 80 degrees Celsius.
Add 100 microliters of STGG medium to the pellet and mix thoroughly, and then determine the total volume in the tube. Next plate, a 20 microliter drop of the resuspended pellet onto a blood agar plate containing gentamycin and streak the entire plate. If the nasal wash is post inoculation, use 10 microliters of the STGG containing the resuspended pellet.
For bacterial quantification by the miles and Misra method, add another 800 microliters of STGG to the nasal wash tube and mix thoroughly. Then plate 25 microliters of the STGG bacterial cell solution onto another blood agar plate and 25 microliters onto a chocolate agar plate, streaking the entirety of both plates. Then freeze the remaining bacteria in two cryo vials at minus 80 degrees Celsius.
Incubate the plates of 37 degrees Celsius overnight in 5%carbon dioxide. The next day, examine the plates for pneumococcus and other potential respiratory pathogens. Blood agar plates, inoculated with nasal washes can be difficult to read.
This image shows an example of a plate that is easy to read with the pneumococci clearly visible. This plate, however, contains many co colonizing flora making it more difficult to determine if pneumococci are present at every appointment. Volunteers were asked to describe any upper respiratory tract symptoms if present and comparison between carriers and non-carriers were highlighted.
Of the 145 people recently inoculated with pneumococcus 28 complained of symptoms as summarized in this table. Eight of these volunteers were carriers, 20 were not. All the complaints were reviewed by the external safety Committee and following full investigations.
No symptoms were attributed to pneumococcal disease. The most common symptoms were sore throat, stuffed nose, and flu-like illness. While attempting this procedure, it's important to have a very thorough safety plan in place, which covers every step of the method, especially for the safety of the volunteers.
After watching this video, you should have a good understanding of how to establish experimental human pneumococcal carriage, and detect carriage using a nasal wash.
