The overall goal of the procedures described with joint aspiration, multiplex polymerase chain reaction and sonication is to deliver fast and reliable microbiological results to the orthopedic surgeons in cases of suspected peri-prosthetic joint infection. This method can help answer key questions in the diagnostic and therapeutic field of peri-prosthetic joint infection, such as early findings of pathogens, and therefore, early targeted treatment. The main advantage of these techniques is the delivery of prompt results.
It can speed up the differential diagnostics in cases of suspected peri-prosthetic joint infection. Position the patient in a supine position on an examination bed. If the knee is to be punctured, place a knee roll under the patient's knee so that the knee rests in a slightly flexed position.
Use electric clippers to shorten dense or long body hair. Then prepare the instrument table and dress for surgery according to the text protocol. Next use a skin disinfectant to thoroughly wash the patient's skin at the puncture site.
With a fenestrated sterile drape, cover puncture site. Then identify the puncture site through the drape. Using a pointed scalpel blade, make a small stab incision through the skin at the incision site.
Attach the cannula to the luer-slip of the syringe. Then insert the cannula through the stab incision. At the superior recess of the knee joint, aim at a 45-degree angle dorsal and medial towards the recess, below the top pole of the patella, and insert the needle to a depth of approximately 5 cm.
Aspirate the joint fluid by drawing out the syringe's piston. To prevent the formation of hematomas, apply a dressing, followed by a bandage to the stab incision. Use alcoholic disinfectant to disinfect the membrane of a pediatric blood culture flask.
Then put a fresh cannula onto the syringe and inject to 1 to 3 mL of aspirated joint fluid into the flask. Mix by gentle agitation or rotation. To prepare a native joint aspirate specimen, remove the cannula from the syringe.
Then open a blood collection tube without additives and inject 1 mL of aspirate into the collection tube. Replace and fasten the lid for PCR analysis. Transfer 1 to 4 mL of the joint aspirate into a blood collection tube containing EDTA for cell count and differentiation.
Defrost the master mix tube by setting it at room temperature. Transfer 180 mcL of the collected specimen into the sample tube. Close the sample tube with the sample tube cap that contains protein kinase K and an internal control gene for the quality control of the entire workflow.
At the cockpit, open the operating software by touching the New Test button in the lower left corner. Use the touch screen to enter the patient's data or a sample ID.To select the specimen from the drop down, first choose ITI Cartridge, then Orthopedics as application mode, and finally, select Synovial Fluid, or Sonication Fluid as sample type. Then press the Start button.
Scan the barcode on the bottom of the sample tube. Then insert the sample tube into the Lysator. Next select the sample on the cockpit screen to unlock the sample tube from the Lysator.
Then remove the sample tube from the Lysator and close the Lysator's top cover. Transfer the sample tube into the sample tube slot of the PCR cartridge. Then insert the defrosted master mix tube into the master mix slot of the PCR cartridge.
Follow the instructions on the cockpit monitor to scan the PCR cartridge with the master mix and sample tube. Insert the PCR cartridge into the indicated cartridge slot of the analyzer. At the cockpit touch screen in the lower left corner, choose Active Tests, then pick the correct sample ID to display the test results.
Save the results on the machine's memory and print or export for further reference. Talk to your microbiologist and brief him on the case details, so he will be prepared to analyze the samples promptly. Under a laminar air-flow working bench, open the plastic container, holding the ex-planted prosthesis from the operating room and add sterile 0.9%sodium chloride solution until the ex-planted foreign body is covered at least 80%Close the plastic container.
Then thoroughly shake or agitate the plastic container for 30 seconds. Transfer the plastic container into the sonication bath. Then check the fluid level in the bath.
Sonicate the specimen for five minutes, with a frequency of 40 plus or minus 2 kilohertz and a power density of 0.22 plus or minus 0.04 watts per square centimeter. Then shake or thoroughly vortex the specimen for 30 seconds. Inside the laminar flow bench, collect 50 mL of the sonication fluid and transfer it to a sterile tube before tightly sealing the tube.
To create a concentrated sonication fluid, centrifuge the tube at 4200 times G for 10 minutes, leaving a residual volume of 10 mL, use the pipette to discard the remaining supernatant. Re-suspend the pellet by pipetting and vortexing. Inoculate each flask of suitable culture media with 0.5 mL of concentrated sonication fluid.
Inoculate the pediatric blood culture flask with 2 mL of concentrated sonication fluid as demonstrated earlier in this video. Then transfer 1 mL of concentrated sonication fluid into a sterile tube without additives for PCR analysis. Transfer the inoculated culture media to the incubator.
Incubate the cultures for 14 days and check for growth daily. The presence of a peri-prosthetic joint infection was considered proven when one major criteria or at least three out of five minor criteria were present. Major criteria include presence of a fistula or detection of a pathogen in two separate microbiological samples.
Minor criteria include positive cell count or positive cell differentiation in joint aspirate, positive CRP and sedimentation rate in blood samples, positive histology in the tissue samples, or detection of a pathogen in just one microbiological sample. The results here showed a moderate sensitivity for PCR diagnostics of sonication fluid and joint aspirate, but a very strong specificity of 100%Whenever PCR showed a positive finding in the joint aspirate or the sonication fluid, the same pathogen was detected later in one of the conventional microbiological cultures. The PCR of the different samples of non-infection cases showed no false-positive results.
Six out of 16 true pathogens in the sonication fluid samples and five out of 13 pathogens from the joint fluid culture were missed by PCR detection. The combined PCR diagnostics of both materials, joint aspirate, and sonication fluid identified 12 out of 18 infection cases correctly. After watching this video, you should have a good understanding of how to improve your diagnostic path in peri-prosthetic joint infection.
You can see how microbiologists and orthopedic surgeons must work together closely to improve differential diagnostics as well as possible. If performed correctly you will have clinically valuable results from the cartridge-based multiplex PCR within five hours after joint aspiration. While attempting this procedure, it is important to remember to work sterile through the entire process.
Don't forget that working with human tissue specimens or microbiological specimens can be extremely hazardous. All precautions, such as personal protection gear, should always be taken while performing this procedure.
