The authors would like to thank Curetis GmbH for supporting this study.

This study was approved by the local ethics committee (046/09, Rev. 3). Informed consent and data privacy statement were obtained from all patients included.
1. Joint Aspiration
NOTE: The procedure should be performed in a surgical theater or an intervention room with comparable aseptic conditions. Assistance by a nurse or doctor&#39;s assistance is helpful but not mandatory.
<ol>
	<li>Position the patient in supine position on an examination bed. Ensure that the joint of interest is free of clothing. Shorten dense or long body hair using electric clippers. Do not remove body hair with a razor. Position a fluoroscope in anteroposterior direction if the hip joint is to be punctured. If the knee is to be punctured, place a knee roll under the patient&#39;s knee such that the knee rests in a slightly flexed position13.</li>
	<li>Prepare an instrument table with the following sterile equipment: sterile swabs, a fenestrated sterile cover drape, a disposable sterile scalpel, pointed surgical blade (type #11), and sterile 10 mL syringe with sterile cannula (20 gauge, 80 mm). Set out alcoholic skin disinfectant, blood collection tubes and a pediatric blood culture flask separately.</li>
	<li>Dress in a surgical gown, hood and mask, and put on sterile gloves.</li>
	<li>Thoroughly wash the patient&#39;s skin at the puncture site (e.g. superior recessus14 for the knee and anterolateral portal area for the hip) with a skin disinfectant. 
	NOTE: Mind the required exposure time for the disinfectant used (usually 90 s at the knee, 120 s at the hip for alcoholic disinfectants).</li>
	<li>Cover the puncture site with a fenestrated sterile cover drape. Identify the puncture site. 
	NOTE: For the knee, the correct site is 2 cm above the upper patella pole and 2 cm lateral to the lateral facet. For the hip, find the anterior superior iliac spine and move caudally until in line with the upper symphysis. Ensure that the site is lateral to the femoral artery (approximately 4 cm), which can always be palpated15.
	<ol>
		<li>Make a small stab incision through the skin (3 - 4 mm wide and deep) with the pointed scalpel blade at the puncture site. Do not apply local anesthetics as they may cause false negatives in the microbiological culture, due to their bacteriostatic effect.</li>
	</ol>
	</li>
	<li>Attach the cannula to the Luer slip of the syringe. Insert the cannula through the stab incision. At the superior recess of the knee joint, aim at a 45&#176; 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&#39;s piston.
	<ol>
		<li>Use fluoroscope guidance when puncturing a hip joint. Insert the needle at the puncture site, aiming medially at a 60&#176; angle. In the fluoroscope, ensure that the tip of the needle is pointed at the prosthesis neck. Advance the cannula to a depth of approximately 8 cm (deeper in adipose patients) while aspirating, until joint fluid is attained. Hold the needle in position while aspirating, to avoid scraping of the prosthesis surface. 
		NOTE: Contact of the needle tip with the prosthesis ensures intraarticular positioning. Usually, no more than 5 mL of fluid can be aspirated form a hip joint, a knee joint will yield more than 10 mL.</li>
		<li>After removal of the cannula, apply a sterile wound dressing to the stab incision. To prevent hematoma formation, apply a bandage to the leg with slight compression.&#160;</li>
	</ol>
	</li>
	<li>Transfer the collected joint aspirate into the sample containers. Ensure sterile working techniques throughout.
	<ol>
		<li>For inoculation of the pediatric blood culture flask with the aspirated joint fluid, disinfect the membrane of the pediatric blood culture flask with alcoholic disinfectant. Put a fresh cannula onto the syringe and inject 1 to 3 mL of aspirated joint fluid into the blood culture flask. Mix by gentle agitation or rotation16.</li>
		<li>For preparation of a native joint aspirate specimen, remove the cannula from the syringe. Open a blood collection tube without additives and inject 1 mL of aspirate into this collection tube. Replace and fasten the lid for PCR analysis. 
		NOTE: Ship the sample to the microbiology lab without delay for PCR analysis. From the same sample, conventional microbiological aerobic and anaerobic cultures can also be set up17.</li>
		<li>Transfer 1 to 4 mL of the joint aspirate into a blood collection tube containing EDTA for cell count and differentiation2. Ship the sample to a biochemistry lab without any delay.</li>
	</ol>
	</li>
</ol>
2. PCR Diagnostics
<ol>
	<li>Make sure all three devices (the lysator, the analyzer, and the cockpit; see the Table of Materials) are up and running correctly. Set out all supplies needed for performing the test (the PCR cartridge, a master mix tube, the sample tube and sample tube cap, a 0 - 200 &#181;L micropipette, and sterile pipette tips) on the working bench.</li>
	<li>Defrost the master mix tube before starting the procedure, by setting it out at room temperature. All consumables (master mix tube, cartridge, and sample tube) are already prelabeled with a barcode.</li>
	<li>Remove the cap of the sample tube. Take 180 &#181;L of the collected specimen (either joint aspirate, see section 1; or sonication fluid, see section 4) with a pipette and transfer it into the sample tube. Close the sample tube with the sample tube cap containing protein kinase K and an internal control gene for the quality control of the entire workflow.</li>
	<li>At the cockpit, open the operating software by touching the &#34;new test&#34; button in the lower left corner. Enter the patient&#39;s data or a sample ID via the touchscreen. To select the specimen, first choose &#34;ITI-Cartridge&#34; from the dropdown, then &#34;Orthopedics&#34; as application mode, and finally select &#34;synovial&#160;fluid&#34; or &#34;sonication fluid&#34; as sample type. Press the start button.</li>
	<li>Scan the barcode on the bottom of the sample tube. Insert the sample tube into the lysator. 
	NOTE: The lysis process will start automatically and take approximately 30 min to finish. Countdown of the timer on the lysator screen will indicate when the lysis is finished.</li>
	<li>Select the sample on the cockpit screen to unlock the sample tube from the lysator. Remove the sample tube from the lysator and close the lysator&#39;s top cover. Transfer the sample tube into the sample tube slot of the PCR cartridge. Insert the defrosted mastermix tube into the mastermix slot of the PCR cartridge.</li>
	<li>Follow the instructions on the cockpit monitor, scanning the PCR cartridge with mastermix and sample tube.</li>
	<li>Insert the PCR cartridge into the indicated cartridge slot of the analyzer. When done, the analyzer releases the cartridge. 
	NOTE: The PCR conditions are preset by the manufacturer and cannot be changed by the user. For further information see the manufacturer&#39;s application guide. The PCR process will start automatically and take approximately 4 h 10 min.</li>
	<li>Remove and discard the cartridge. At the cockpit touchscreen, choose &#34;current tests&#34; in the lower left corner, then pick the correct sample ID to display the test results. Save the results on the machine&#39;s memory, and print or export (via USB drive) for further reference. 
	NOTE: Report the result of the PCR, including pathogen identification and detection of resistance markers, to the surgeon without delay. A panel with 114 deoxyribonucleic acid (DNA) targets of bacterial and fungal pathogens typically found in implant infections and soft tissue infections, along with the most common antibiotic resistance markers is analyzed.</li>
</ol>
3. Surgical Procedure: Intraoperative Sample Collection
NOTE: Revision arthroplasty surgery requires an expert level of skill and expertise; for a comprehensive overview of the surgical procedures, please refer to surgeon&#39;s textbooks18. A full and detailed description of the surgical procedure would be well beyond the scope of this article. Here the focus is on the details of the sample collection and pre-analytics. In revision arthroplasty surgery, refrain from administration of a single shot antibiotic prophylaxis, as not to compromise the results of the microbiological samples obtained during surgery. Adhering to this rule is recommended, though this practice is controversial19,20. Use the existing surgical approach to the joint whenever possible. Additional surgical approaches will compromise soft tissues and increase scar formation.
<ol>
	<li>Dissect tissue until the joint capsule is well exposed. Perform the arthrotomy with a syringe at the ready, so further joint fluid can be collected into the sterile syringe for further analysis as stated above (steps 1.7.1-1.7.3). 
	NOTE: No antiseptic lavage fluids must be used until the implant is removed and tissue samples have been taken.</li>
	<li>Remove the joint implants. Immediately after removal, transfer the implant parts into a sterile plastic container with an air- and water-tight lid. 
	NOTE: Detailed instructions for implant removal can be found in the textbook literature (Knee, e.g.: Wirtz et al., Chapter 16.421; Hip, e.g.: Claes et al., Chapter 14.5.122). Specific instruments may be necessary, as stated by the manufacturers&#39; instructions. The implant removal technique varies considerably between different implant types and fixation methods. A set of chisels and drills, a sliding hammer and a universal intramedullary nail removal set are helpful in removing bony integrated implants23,24.</li>
	<li>Use a sterile sharp curette, forceps and rongeur to remove the membrane tissue from the bone-implant interface. Transfer a representative sample of the tissue into a sterile plastic container with a screw-on lid, as specimens for microbiology and histology. 
	NOTE: A reamer can be used to clear the medullary canal of all soft tissues. Also, take synovial tissue and articular capsule samples for examination and store them in sterile containers. At least four specimens in total must be gathered.</li>
	<li>Send all samples and explanted parts to the microbiology lab instantly. 
	NOTE: Antibiotics can then be given. The choice of the correct antibiotics is essential and must be an individualized decision25,26. Therapeutic concepts must be decided on by an interdisciplinary panel of surgeons and microbiologists beforehand.</li>
	<li>Complete the debridement of the former implant site by removing any tissue that shows signs of debris (such as polyethylene wear, metal or cement particles) or infection and necrosis (purulent, avascular, fibrin-coated, membranous or &#34;sludgy&#34; tissues). Remove any dead or osteitic bone. Remove all foreign material such as screws, wires, cerclages, bone cement debris or non-resorbable sutures (see Claes et al., Chapter 14.5.322).</li>
	<li>Irrigate the surgical site using a wound disinfectant of choice using a 50-mL syringe. Irrigate with ample amounts (at least 3 L) of ringer solution with a pulsed lavage system. A spacer may be necessary to stabilize the joint27.</li>
	<li>Close the wound using antimicrobial-coated resorbable deep sutures for the capsule or fascia (braided polyglactin sutures, triclosan-coated, USP 2), and subcutaneous tissue (braided polyglactin sutures, triclosan-coated, USP 0). Close the skin using non-resorbing interrupted sutures (monofilic polypropylene, USP 0 or USP 2-0).</li>
</ol>
4. Sonication
NOTE: Be careful to work strictly aseptically. Use a Class II biosafety bench with laminar air-flow for further processing of the explanted material.
<ol>
	<li>Open the plastic container holding the explanted prosthesis from the operating room (see step 3.2) under a laminar air-flow working bench, and add sterile 0.9% sodium chloride solution until the explanted foreign body is covered at least 80% (approx. 500-800 mL).</li>
	<li>Close the plastic container. Shake thoroughly or agitate the plastic container using a vortex mixer on high intensity for 30 s. Transfer the plastic container into the sonication bath. Check the fluid level in the sonication bath. 
	NOTE: The sonication bath fluid level must be the same as inside the plastic container. Ensure that the plastic container cannot be flooded and if necessary add or remove liquid from the sonication bath.</li>
	<li>Sonicate the specimen for 5 min with a frequency of 40 &#177; 2 kHz and power density 0.22 &#177; 0.04 W/cm2. Shake or vortex the specimen thoroughly for 30 s. 
	NOTE: Sonication times between 1-5 min are best for dissolving biofilms, without any influence on bacterial viability28.</li>
	<li>Inside the laminar flow bench, collect 50 mL of the sonication fluid and transfer it to a sterile, tightly-sealed tube.</li>
	<li>To create a concentrated sonication fluid, centrifuge the tube for 10 min at 4,200 x g. Leaving a residual volume of 10 mL, discard the remaining supernatant with a pipette. Resuspend the pellet by pipetting and vortexing.</li>
	<li>Inoculate suitable culture media (e.g. blood agar, chocolate agar, Sabouraud agar, Schaedler&#39;s agar, Kanamycin-Vancomycin agar, or thioglycolate broth) with 0.5 mL of concentrated sonication fluid each. Inoculate the pediatric blood culture flask with 2 mL as described above (see step 1.7.1). Transfer 1 mL of concentrated sonication fluid into a blood collection tube without additives for PCR analysis.</li>
	<li>Transfer the inoculated culture media to the incubator (35 &#176;C, 5% CO2). Incubate the cultures for 14 days, and check for growth daily.</li>
	<li>After 14 days, discard the cultures with no growth and report as negative. If growth is detected, carry out pathogen identification using standard identification techniques and susceptibility testing. Report the result to the surgeon without delay. 
	NOTE: Here, identification of pathogens was carried out using Matrix-assisted LASER desorbtion/ionization - time of flight (MALDI-TOF) spectroscopy. Antimicrobial susceptibility testing was performed with a semiautomated analyzer29,30,31.</li>
</ol>

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E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advantages+of+sonication+fluid+culture+for+the+diagnosis+of+prosthetic+joint+infection.">Advantages of sonication fluid culture for the diagnosis of prosthetic joint infection.</a> J Infect. 69 (1), 35-41 (2014).</li><li>Scorzolini, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sonication+technique+improves+microbiological+diagnosis+in+patients+treated+with+antibiotics+before+surgery+for+prosthetic+joint+infections.">Sonication technique improves microbiological diagnosis in patients treated with antibiotics before surgery for prosthetic joint infections.</a> New Microbiol. 37, 321-328 (2014).</li><li>Zhai, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meta-analysis+of+sonication+fluid+samples+from+prosthetic+components+for+diagnosis+of+infection+after+total+joint+arthroplasty.">Meta-analysis of sonication fluid samples from prosthetic components for diagnosis of infection after total joint arthroplasty.</a> J Clin Microbiol. 52 (5), 1730-1736 (2014).</li><li>Panousis, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poor+predictive+value+of+broad-range+PCR+for+the+detection+of+arthroplasty+infection+in+92+cases.">Poor predictive value of broad-range PCR for the detection of arthroplasty infection in 92 cases.</a> Acta Orthop. 76 (3), 341-346 (2005).</li><li>Rak, M., KavcIc, M., Trebse, R., Co, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+bacteria+with+molecular+methods+in+prosthetic+joint+infection:+sonication+fluid+better+than+periprosthetic+tissue.">Detection of bacteria with molecular methods in prosthetic joint infection: sonication fluid better than periprosthetic tissue.</a> Acta Orthop. 87 (4), 339-345 (2016).</li><li>Li, Z., Yu, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+value+of+a+PCR-based+technique+for+prosthetic+joint+infection.">Diagnostic value of a PCR-based technique for prosthetic joint infection.</a> J Clin Microbiol. 52 (6), 2281-2282 (2014).</li></ol>

The authors have nothing to disclose.

Foreign body infection is an emerging problem in orthopedic and trauma surgery with costly treatment, long hospitalization and functional deficiency of the affected joints. The differential diagnostic is challenging. Many researchers and clinicians are giving attention to this topic, striving to find more precise and reliable methods to diagnose foreign body associated infections. To date, many different diagnostic tools are being evaluated and implemented in the diagnostic path32.
The sonication procedure is an invaluable method, showing a better sensitivity than conventional cultures from tissue specimens6. In our study, we showed that sonication fluid cultures have a sensitivity of 88.9% with a specificity of 61.5%. Conventional cultures from tissue specimens and joint aspirates both had a sensitivity of 66.7% with a specificity of 82.3% and 84.6%, respectively. Other researchers show similar results for these conventional microbiological procedures6,33,34,35. It is often criticized that sonication procedure is prone to contamination, so special care must be taken in the handling of the samples.
The possibility of detecting DNA of a causative pathogen in tissue specimens or fluids is intriguing. NAT is a rapid procedure which can deliver results within a couple of hours, as compared to the time-consuming microbiological culture methods. Without doubt, NAT has a good sensitivity in detecting pathogens, but hold the risk of detecting contaminants, thus lacking specificity36. The great benefit of this PCR technique in diagnosing PJI was documented especially in patients who received antibiotic therapy close to surgery or for a longer period7,8. Studies suggest that NAT of sonication fluid may further increase sensitivity and specificity37. Unfortunately, this technique is not routinely available in laboratories, due to its time-consuming workflow.
There are certain limitations to the PCR technique in general: NAT detects DNA with no differentiation between viable and non-viable bacteria, making interpretation of the results difficult. Broad-range PCR will only detect ribosomal 16S ribonucleic acid (16S rRNA), not differentiating between pathogens37. More specific systems do not routinely detect gene-encoded antibiotic resistance markers. A targeted antibiotic therapy may therefore be limited without further susceptibility testing.
The system applied in this study overcomes some of these limitations, differentiating between specific bacteria and identifying gene-encoded resistance markers. Overall, NAT assays may be considered as a fast and useful complementation to confirm PJI7,8,9,38.
It is necessary to plan ahead in joint aspiration and in surgery: Sample containers must be sterile and ready, and prompt sample transport must be available. Surgeons should brief their microbiologists on planned procedures and what sample material to expect. When performing the joint aspiration, strictly sterile conditions must be ensured, to prevent iatrogenic infection of the joint. In adipose patients, joint puncture can be challenging, and fluoroscopic guidance can be helpful. Revision arthroplasty surgery requires a very high level of expertise, and should only be performed by a skilled surgeon. Explanted material should not be kept in the operating room any longer than necessary, but be sent to the microbiologist as soon as possible. A very critical part within this protocol is the potential risk of contamination. Not only while gathering the samples, but also while handling the samples in the microbiology lab, all personnel involved (orthopedic surgeon, nurses, technicians, microbiologists) must work quickly and accurately, and have proper training in the procedures.
Sonication and NAT are valuable tools in diagnosing implant-associated infections. Nevertheless, the results should always be questioned carefully. We recommend discussing the results in a round table discussion of orthopedic surgeons, microbiologists and infectious disease specialists, and pathologists to agree on the individualized therapeutic strategy.
To implement this technique in the diagnostic path of PJI can have several advantages. It is a rapid diagnostic with a result within hours. Due to the analysis of several gene encoded resistance markers, a targeted antibiotic therapy can take place at a very early stage in the clinical course. As a side effect, broad-range antibiotics can be saved for the indications where they are truly needed. Other sample types (e.g. tissue specimens, swabs, hematoma, etc.) can also be investigated according to our protocol. Since the PCR cartridge is a closed system, no troubleshooting is necessary or possible on the user side. Any adaptation of the protocol must be implemented by the manufacturer. Changes in both software and hardware (cartridge) are continuously made by the manufacturer to enhance the system, however no details are made public on the exact changes in newer versions.

Painful arthroplasties are a diagnostic challenge in orthopedic surgery. After aseptic loosening, PJI is the second most reason for implant failure, and presents a devastating complication after arthroplasty surgery. PJI is difficult to treat and difficult to diagnose. Missed diagnosis of a PJI will likely result in recurrent infection, with major morbidity, loss of function, and loss of quality of life. Therefore, infection should be ruled out in all patients presenting with painful arthroplasty, before therapeutic measures are initiated.
Patient history, clinical examination, blood examination for CRP and white blood cell count, as well as radiography or szintigraphy of the affected joint constitute the basic diagnostics1. The preoperative routine should also include a joint aspiration under sterile conditions wherever possible. The aspirate acquired is a very valuable material in further diagnostics.
Besides the cell count and cell differentiation in joint aspirate as well-established assays2, the detection of protein biomarkers may help in differential diagnostics3,4,5. Conventional microbiological culture remains the gold standard in pathogen detection. Biofilms, caused by both gram-positive and gram-negative bacteria, and adherent to the implant surfaces, are a major pathogenic factor in PJI. Therefore, in 2007, the sonication procedure was implemented in the diagnostics of foreign body-associated infections in orthopedic surgery, disrupting the biofilms on removed implants to allow pathogen detection. The bacteria are thereby taken from their resting form to an active form, making detection in culture possible again. Sonication of removed implants showed a higher sensitivity than cultures from tissue specimens (78.5% vs. 60.8%)6.
Nucleic acid amplification test (NAT), such as PCR, has recently moved into the scope of clinicians and microbiologists to diagnose PJI. Especially in patients who received antibiotic therapy prior to surgery, it was shown that the PCR diagnostic is beneficial in identifying the causative organisms7,8,9. Lately, a new multiplex PCR system, specially designed for implant and tissue infections (ITI), was introduced. This cartridge-based system offers a variety of genomic markers for identification of pathogens and antibiotic resistance markers. Among its application range are numerous indications (prosthetic joint infections, surgical site infections, cardiology-related infections, catheter-associated infections, diabetic foot infections, deep skin and tissue infections, implant infections, and burn-wound infections), and a broad panel of sample material can be used for this technique (sonication fluid, synovial fluid, swabs, tissue, pus, aspirate/exudate, and biofilm)10,11,12.
The greatest advantage of the procedure, as compared to conventional microbiology, is speed: The causative pathogen can be identified within hours. Additionally, this PCR system detects a broad panel of gene-encoded resistance markers, allowing the initiation of targeted antibiotic therapy early on. With the assistance of PCR, surgeons may be able to distinguish between infected and non-infected patients at a very early stage in the diagnostic procedure11. Here, we present the protocol to perform this multiplex PCR, quickly diagnosing PJI from joint aspirate and sonication fluid.

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			<td height="42" style="height:42px;width:216px;">sterile plastic container&#160;</td>
			<td style="width:333px;">Lock &#38; Lock, Distributor ISI GmbH, Solingen, Germany</td>
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			<td>Bactosonic, Bandelin, Berlin, Germany</td>
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			<td height="21" style="height:21px;width:216px;">50ml Falcon tubes</td>
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			<td height="21" style="height:21px;">PEDS medium blood culture flasks&#160;</td>
			<td>Becton &#38; Dickinson, Heidelberg, Germany</td>
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			<td>culture medium</td>
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			<td height="21" style="height:21px;">Columbia agar with 5% sheep blood</td>
			<td>Becton &#38; Dickinson, Heidelberg, Germany</td>
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			<td>culture medium</td>
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			<td>culture medium</td>
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			<td>Becton &#38; Dickinson, Heidelberg, Germany</td>
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			<td>culture medium</td>
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			<td height="21" style="height:21px;">sabouraud agar&#160;</td>
			<td>Becton &#38; Dickinson, Heidelberg, Germany</td>
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			<td>culture medium</td>
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			<td height="21" style="height:21px;">thioglycolate bouillon&#160;</td>
			<td>Becton &#38; Dickinson, Heidelberg, Germany</td>
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			<td>culture medium</td>
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			<td height="21" style="height:21px;">Schaedler agar</td>
			<td>Becton &#38; Dickinson, Heidelberg, Germany</td>
			<td align="right" style="width:119px;">254084</td>
			<td>culture medium</td>
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			<td height="21" style="height:21px;">kanamycin/vancomycin agar</td>
			<td>Becton &#38; Dickinson, Heidelberg, Germany</td>
			<td align="right" style="width:119px;">254077</td>
			<td>culture medium</td>
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			<td height="21" style="height:21px;">Bactec FX blood culture system&#160;</td>
			<td>Becton &#38; Dickinson, Heidelberg, Germany</td>
			<td style="width:119px;">441386/441385</td>
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			<td height="21" style="height:21px;width:216px;">Unyvero A50 Analyzer</td>
			<td style="width:333px;">Curetis, Holzgerlingen, Germany</td>
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			<td>PCR machine</td>
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			<td height="21" style="height:21px;width:216px;">Unyvero L4 Lysator</td>
			<td style="width:333px;">Curetis, Holzgerlingen, Germany</td>
			<td align="right" style="width:119px;">60002</td>
			<td>PCR machine</td>
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			<td height="21" style="height:21px;width:216px;">Unyvero C8 Cockpit</td>
			<td style="width:333px;">Curetis, Holzgerlingen, Germany</td>
			<td align="right" style="width:119px;">60003</td>
			<td>PCR machine</td>
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			<td height="21" style="height:21px;width:216px;">Unyvero M1 Masterix Tube</td>
			<td style="width:333px;">Curetis, Holzgerlingen, Germany</td>
			<td align="right" style="width:119px;">10002</td>
			<td>consumables PCR</td>
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			<td height="21" style="height:21px;width:216px;">Unyvero i60 ITI Cartridge</td>
			<td style="width:333px;">Curetis, Holzgerlingen, Germany</td>
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			<td>consumables PCR</td>
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			<td height="21" style="height:21px;width:216px;">Unyvero Sample Tube Cap</td>
			<td style="width:333px;">Curetis, Holzgerlingen, Germany</td>
			<td align="right" style="width:119px;">10004</td>
			<td>consumables PCR</td>
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			<td height="21" style="height:21px;width:216px;">Unyvero Sample Tube</td>
			<td style="width:333px;">Curetis, Holzgerlingen, Germany</td>
			<td align="right" style="width:119px;">10003</td>
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			<td height="21" style="height:21px;width:216px;">S-Monovette 2.7ml</td>
			<td style="width:333px;">Sarstedt AG, N&#252;mbrecht, Germany</td>
			<td style="width:119px;">05.1729.001&#160;</td>
			<td>Transport container (aspirate)</td>
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			<td height="21" style="height:21px;width:216px;">scalpel typ 11</td>
			<td style="width:333px;">pfm medical, Cologne, Germany</td>
			<td align="right">200130011</td>
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			<td height="21" style="height:21px;width:216px;">PP Container 70mL 55x45mm</td>
			<td style="width:333px;">Sarstedt AG, N&#252;mbrecht, Germany</td>
			<td align="right">759,922,721</td>
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			<td height="42" style="height:42px;width:216px;">Vicryl Plus</td>
			<td style="width:333px;">Johnson&#38;Johnson Medical GmbH, Ethicon Germany, Norderstedt, Germany</td>
			<td>VCP247H</td>
			<td>surgical suture material</td>
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			<td height="42" style="height:42px;width:216px;">Prolene 0</td>
			<td style="width:333px;">Johnson&#38;Johnson Medical GmbH, Ethicon Germany, Norderstedt, Germany</td>
			<td>EH7920H</td>
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			<td height="42" style="height:42px;width:216px;">Prolene 2/0</td>
			<td style="width:333px;">Johnson&#38;Johnson Medical GmbH, Ethicon Germany, Norderstedt, Germany</td>
			<td>EH7697H</td>
			<td>surgical suture material</td>
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			<td height="21" style="height:21px;width:216px;">Kodan&#160; Tinktur forte farblos</td>
			<td style="width:333px;">Sch&#252;lke &#38; Mayr GmbH, Norderstedt, Germany</td>
			<td align="right" style="width:119px;">104005</td>
			<td>alcoholic skin disinfectant</td>
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novel diagnostics in revision arthroplasty: implant sonication and multiplex polymerase chain reaction

In orthopedic patients, foreign body-associated infections, especially periprosthetic joint infections (PJIs), are a devastating complication of arthroplasty. Infection requires complex treatment, may result in long hospitalization and causes considerable costs. Multiple surgical revisions can be necessary in these patients, with a loss in function as well as in quality of life.
The routine preoperative diagnostics include blood examination for C-reactive protein (CRP) and other biomarkers, as well as joint aspirate analysis for cell count, differentiation, and culture. Intraoperative specimens for histology and microbiology are also standard procedure. The microbiological examination of removed implants with sonication, in combination with the implementation of molecular biology techniques in microbiology, represent two novel techniques currently employed to enhance the differential diagnostics of PJI.
We present here the step-wise procedure of analyzing joint aspirate and sonication fluid, using a cartridge-based multiplex polymerase chain reaction (PCR) system. Results were matched against conventional cultures and consensus criteria for PJI. Conventional microbiological cultures from tissue biopsies, joint aspirate and sonication fluid showed a sensitivity of 66.7%, 66.7%, and 88.9%, respectively, and a specificity of 82.3%, 54.6%, and 61.5%, respectively. The PCR diagnostic of the sonication fluid and the joint fluid showed a sensitivity of 50.0% and 55.6%, respectively, and both a specificity of 100.0%. Both PCR diagnostics combined had a sensitivity of 66.7% and a specificity of 100.0%. The multiplex PCR therefore presents a rapid diagnostic tool with moderate sensitivity but high specificity in diagnosing PJI.

Presence of a PJI, as defined by the consensus meeting of the Musculoskeletal Infection Society (MSIS), 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 ( &#62;3,000 leukocytes/&#181;L) or positive cell differentiation ( &#62;85% neutrophil granulocytes) 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 sample19. Sensitivity and specificity were calculated for nucleic acid amplification test (NAT), as compared to the MSIS gold standard. Patient details, including pathogens detected, are given in Table 1.
Our own results showed a moderate sensitivity for PCR diagnostic of sonication fluid and joint aspirate (50.0% and 55.6%) but a very strong specificity of 100%11. Whenever PCR diagnostic (total n = 62 tests) showed a positive finding in the joint aspirate (n = 31) or the sonication fluid (n = 31), 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 result (see Figure 1).
The PCR diagnostic failed to detect some pathogens that were proven with conventional microbiological culture methods. 6 out of 16 (37.0%) true pathogens in the sonication fluid samples and 5 out of 13 (38.0%) pathogens from the joint fluid culture were missed by PCR detection. Mostly, the PCR diagnostics missed detection of coagulase-negative staphylococci (8 out of 11). The combined PCR diagnostics of both materials (joint aspirate and sonication fluid, &#34;pooled PCR&#34;) identified 12 out of 18 infection cases correctly (sensitivity 66.7%, 95% CI: 41.0% to 86.7%, see Table 2).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55147/55147fig1.jpg" /> 
Figure 1: Summary of NAT results. The graph depicts the summarized results from the collective patient samples. On the right side is the group of patients matching PJI criteria, on the left side is the group that did not. The bars represent the nucleic acid amplification methods. False positives and false negatives are shown in red as percentage of the total. <a href="http://cloudflare.jove.com/files/ftp_upload/55147/55147fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Joint</td>
			<td>Cause of Revision</td>
			<td>Matches MSIS Criteria?</td>
			<td>Previous antibiotic treatment?</td>
			<td>Sonication fluid culture</td>
			<td>Joint aspirate culture</td>
			<td>NAT sonication culture</td>
			<td>NAT joint aspirate</td>
		</tr>
		<tr>
			<td>Hip</td>
			<td>aseptic loosening</td>
			<td>No</td>
			<td>No</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Knee</td>
			<td>aseptic loosening</td>
			<td>No</td>
			<td>No</td>
			<td>Staphlococcus 
			epidermidis</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Knee</td>
			<td>aseptic loosening</td>
			<td>No</td>
			<td>No</td>
			<td>Dermabacter 
			hominis</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Knee</td>
			<td>aseptic loosening</td>
			<td>No</td>
			<td>No</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Knee</td>
			<td>aseptic loosening</td>
			<td>No</td>
			<td>No</td>
			<td>Leifsonia 
			aquatica</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hip</td>
			<td>aseptic loosening</td>
			<td>No</td>
			<td>No</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Knee</td>
			<td>aseptic loosening</td>
			<td>No</td>
			<td>No</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Knee</td>
			<td>instability</td>
			<td>No</td>
			<td>No</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hip</td>
			<td>chronic luxation</td>
			<td>No</td>
			<td>No</td>
			<td>Staphylococcus 
			haemolyticus</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Knee</td>
			<td>aseptic loosening</td>
			<td>No</td>
			<td>No</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hip</td>
			<td>aseptic loosening</td>
			<td>No</td>
			<td>No</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hip</td>
			<td>aseptic loosening</td>
			<td>No</td>
			<td>No</td>
			<td>Staphlococcus 
			epidermidis</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Knee</td>
			<td>instability</td>
			<td>No</td>
			<td>No</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Knee</td>
			<td>acute infection</td>
			<td>Yes</td>
			<td>Yes</td>
			<td>Pseudomonas aeruginosa</td>
			<td>Pseudomonas aeruginosa</td>
			<td></td>
			<td>Pseudomonas 
			aeruginosa</td>
		</tr>
		<tr>
			<td>Knee</td>
			<td>acute infection</td>
			<td>Yes</td>
			<td>No</td>
			<td>Escherichia 
			coli</td>
			<td>Escherichia 
			coli</td>
			<td>Escherichia 
			coli</td>
			<td></td>
		</tr>
		<tr>
			<td>Hip</td>
			<td>chronic infection</td>
			<td>Yes</td>
			<td>No</td>
			<td>Corynebakterium 
			spp.</td>
			<td>Staphlococcus 
			epidermidis</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Knee</td>
			<td>acute infection</td>
			<td>Yes</td>
			<td>No</td>
			<td>Staphylococcus aureus</td>
			<td>Staphylococcus aureus</td>
			<td>Staphylococcus aureus</td>
			<td>Staphylococcus 
			aureus</td>
		</tr>
		<tr>
			<td>Knee</td>
			<td>chronic infection</td>
			<td>Yes</td>
			<td>No</td>
			<td>Streptococcus 
			agalacticae</td>
			<td>Streptococcus 
			agalacticae</td>
			<td>Streptococcus 
			agalacticae</td>
			<td>Streptococcus 
			agalacticae</td>
		</tr>
		<tr>
			<td>Hip</td>
			<td>chronic infection</td>
			<td>Yes</td>
			<td>Yes</td>
			<td></td>
			<td>Staphylococcus aureus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Knee</td>
			<td>chronic infection</td>
			<td>Yes</td>
			<td>No</td>
			<td>Staphlococcus 
			epidermidis</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Knee</td>
			<td>chronic infection</td>
			<td>Yes</td>
			<td>Yes</td>
			<td>Staphlococcu 
			&#160;epidermidis</td>
			<td>Staphlococcus 
			epidermidis</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hip</td>
			<td>chronic infection</td>
			<td>Yes</td>
			<td>No</td>
			<td>Staphlococcus 
			epidermidis</td>
			<td>Staphlococcus 
			epidermidis</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Knee</td>
			<td>chronic infection</td>
			<td>Yes</td>
			<td>No</td>
			<td>Staphlococcus 
			epidermidis</td>
			<td></td>
			<td>Coagulase-negative 
			Staphylococci</td>
			<td>Coagulase-negative 
			Staphylococci</td>
		</tr>
		<tr>
			<td>Hip</td>
			<td>chronic infection</td>
			<td>Yes</td>
			<td>No</td>
			<td>Staphylococcus aureus</td>
			<td></td>
			<td>Staphylococcus aureus</td>
			<td></td>
		</tr>
		<tr>
			<td>Knee</td>
			<td>acute infection</td>
			<td>Yes</td>
			<td>No</td>
			<td>Staphlococcus 
			epidermidis</td>
			<td>Staphlococcus 
			epidermidis</td>
			<td>Coagulase-negative 
			Staphylococci</td>
			<td>Coagulase-negative 
			Staphylococci</td>
		</tr>
		<tr>
			<td>Hip</td>
			<td>acute infection</td>
			<td>Yes</td>
			<td>No</td>
			<td></td>
			<td>Staphlococcus 
			epidermidis</td>
			<td></td>
			<td>Coagulase-negative 
			Staphylococci</td>
		</tr>
		<tr>
			<td>Hip</td>
			<td>chronic infection</td>
			<td>Yes</td>
			<td>No</td>
			<td>Staphlococcus 
			epidermidis</td>
			<td>Staphlococcus 
			epidermidis</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hip</td>
			<td>acute infection</td>
			<td>Yes</td>
			<td>No</td>
			<td></td>
			<td>Staphlococcus 
			epidermidis</td>
			<td>Coagulase-negative 
			Staphylococci</td>
			<td></td>
		</tr>
		<tr>
			<td>Knee</td>
			<td>chronic infection</td>
			<td>Yes</td>
			<td>No</td>
			<td>Staphylococcus aureus</td>
			<td>Staphylococcus aureus</td>
			<td></td>
			<td>Staphylococcus 
			aureus</td>
		</tr>
		<tr>
			<td>Hip</td>
			<td>chronic infection</td>
			<td>Yes</td>
			<td>No</td>
			<td>Enterococcus 
			faecialis</td>
			<td>Enterococcus 
			faecialis</td>
			<td>Enterococcus 
			spp.</td>
			<td>Enterococcus 
			spp.</td>
		</tr>
		<tr>
			<td>Knee</td>
			<td>chronic infection</td>
			<td>Yes</td>
			<td>Yes</td>
			<td>Enterocuccus 
			faecalis</td>
			<td>Enterocuccus 
			faecalis</td>
			<td>Enterococcus 
			spp.</td>
			<td>Enterococcus 
			spp.</td>
		</tr>
	</tbody>
</table>
Table 1: Patient details and detected pathogens. Tabular results of the patient details, including the cause of revision surgery, the MSIS criteria matching and the detected pathogens in conventional microbiology and NAAT analysis.
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Criteria</td>
			<td>PCR Sonicate</td>
			<td>PCR Aspirate</td>
			<td>Pooled PCR</td>
		</tr>
		<tr>
			<td>P value</td>
			<td>0.0036</td>
			<td>0.0013</td>
			<td>0.0001</td>
		</tr>
		<tr>
			<td>Sensitivity</td>
			<td>50.0%</td>
			<td>55.6%</td>
			<td>66.7%</td>
		</tr>
		<tr>
			<td>Specificity</td>
			<td>100.0%</td>
			<td>100.0%</td>
			<td>100.0%</td>
		</tr>
		<tr>
			<td>Positive Predictive Value</td>
			<td>100.0%</td>
			<td>100.0%</td>
			<td>100.0%</td>
		</tr>
		<tr>
			<td>Negative Predictive Value</td>
			<td>59.1%</td>
			<td>61.9%</td>
			<td>68.4%</td>
		</tr>
	</tbody>
</table>
Table 2: Results of microbiological methods. Tabular results of the evaluation of the PCR diagnostic of sonication fluid, joint aspirate and pooled PCR. N = 31 specimen for aspirate and sonication, respectively, n = 62 for the pooled PCR data.

Watch this Scientific Journal Video about Novel Diagnostics in Revision Arthroplasty: Implant Sonication and Multiplex Polymerase Chain Reaction at JoVE.com

Novel Diagnostics in Revision Arthroplasty: Implant Sonication and Multiplex Polymerase Chain Reaction

Differential diagnostics in painful arthroplasty is crucial for treatment success. Joint aspiration is routinely performed preoperatively. Multiplex polymerase chain reaction of the joint aspirate and the sonication fluid, a feasible tool for quick pathogen detection from these samples is described.

In orthopedic patients, foreign body-associated infections, especially periprosthetic joint infections (PJIs), are a devastating complication of ...

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11.0K Views.  University Hospital Bonn. Differential diagnostics in painful arthroplasty is crucial for treatment success. Joint aspiration is routinely performed preoperatively. Multiplex polymerase chain reaction of the joint aspirate and the sonication fluid, a feasible tool for quick pathogen detection from these samples is described.

Video: Novel Diagnostics in Revision Arthroplasty: Implant Sonication and Multiplex Polymerase Chain Reaction

Reverse Total Shoulder Arthroplasty

Reverse total shoulder arthroplasty was initially approved for use in rotator cuff arthropathy and well as chronic pseudoparalysis without arthritis in patients who were not appropriate for tendon transfer reconstructions. Traditional surgical options for these patients were limited and functional results were sub-optimal and at times catastrophic. The use of reverse shoulder arthroplasty has been found to effectively restore these patients function and relieve symptoms associated with their disease. The procedure can be done through two approaches, the deltopectoral or the superolateral. Complication rates associated with the use of the prosthesis have ranged from 8-60% with more recent reports trending lower as experienced is gained. Salvage options for a failed reverse shoulder prosthesis are limited and often have significant associated disability. Indications for the use of this prosthesis continue to be evaluated including its use for revision arthroplasty, proximal humeral fracture and tumor. Careful patient selection is essential because of the significant risks associated with the procedure.

Reverse total shoulder arthroplasty is indicated for the treatment of conditions that cannot be treated with conventional arthroplasty or other procedures. These primarily include degenerative and incapacitating conditions with irreparable rotator cuff and loss of the normal biomechanical coupling of the shoulder.

Reverse total shoulder arthroplasty was initially approved for use in rotator cuff arthropathy and well as chronic pseudoparalysis without arthritis in ...

RNAscope for In situ Detection of Transcriptionally Active Human Papillomavirus in Head and Neck Squamous Cell Carcinoma

The &#39;gold standard&#39; for oncogenic HPV detection is the demonstration of transcriptionally active high-risk HPV in tumor tissue. However, detection of E6/E7 mRNA by quantitative reverse transcription polymerase chain reaction (qRT-PCR) requires RNA extraction which destroys the tumor tissue context critical for morphological correlation and has been difficult to be adopted in routine clinical practice. Our recently developed RNA in situ hybridization technology, RNAscope, permits direct visualization of RNA in formalin-fixed, paraffin-embedded (FFPE) tissue with single molecule sensitivity and single cell resolution, which enables highly sensitive and specific in situ analysis of any RNA biomarker in routine clinical specimens. The RNAscope HPV assay was designed to detect the E6/E7 mRNA of seven high-risk HPV genotypes (HPV16, 18, 31, 33, 35, 52, and 58) using a pool of genotype-specific probes. It has demonstrated excellent sensitivity and specificity against the current &#39;gold standard&#39; method of detecting E6/E7 mRNA by qRT-PCR. HPV status determined by RNAscope is strongly prognostic of clinical outcome in oropharyngeal cancer patients.

RNAscope for In situ Detection of Transcriptionally Active Human Papillomavirus in Head and Neck Squamous Cell Carcinoma

The presence of high-risk HPV in head and neck tumor tissue is associated with favorable outcomes. The recently developed RNA in situ hybridization technique called RNAscope allows direct visualization of HPV E6/E7 mRNA in FFPE tissue sections.

The &#39;gold standard&#39; for oncogenic HPV detection is the demonstration of transcriptionally active high-risk HPV in tumor tissue. However, detection ...

Combined In vivo Optical and &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in vivo optical and &#956;CT imaging allow the visualization of biological phenomena in an anatomical context. These imaging modalities may be especially useful to study conditions that impact bone. In particular, orthopaedic implant infections are an important problem in clinical orthopaedic surgery. These infections are difficult to treat because bacterial biofilms form on the foreign surgically implanted materials, leading to persistent inflammation, osteomyelitis and eventual osteolysis of the bone surrounding the implant, which ultimately results in implant loosening and failure. Here, a mouse model of an infected orthopaedic prosthetic implant was used that involved the surgical placement of a Kirschner-wire implant into an intramedullary canal in the femur in such a way that the end of the implant extended into the knee joint. In this model, LysEGFP mice, a mouse strain that has EGFP-fluorescent neutrophils, were employed in conjunction with a bioluminescent Staphylococcus aureus strain, which naturally emits light. The bacteria were inoculated into the knee joints of the mice prior to closing the surgical site. In vivo bioluminescent and fluorescent imaging was used to quantify the bacterial burden and neutrophil inflammatory response, respectively. In addition, &#956;CT imaging was performed on the same mice so that the 3D location of the bioluminescent and fluorescent optical signals could be co-registered with the anatomical &#956;CT images. To quantify the changes in the bone over time, the outer bone volume of the distal femurs were measured at specific time points using a semi-automated contour based segmentation process. Taken together, the combination of in vivo bioluminescent/fluorescent imaging with &#956;CT imaging may be especially useful for the noninvasive monitoring of the infection, inflammatory response and anatomical changes in bone over time.

Combined In vivo Optical and  &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Combined optical and &#956;CT imaging in a mouse model of orthopaedic implant infection, utilizing a bioluminescent engineered strain of Staphylococcus aureus, provided the capability to noninvasively and longitudinally monitor the dynamics of the bacterial infection, as well as the corresponding inflammatory response and anatomical changes in the bone.

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in ...

Individualized Stem-positioning in Calcar-guided Short-stem Total Hip Arthroplasty

Bone- and soft-tissue sparing short stems are increasingly used in total hip arthroplasty (THA). However, there are a large variety of models of short stems, differing in design and function. Calcar-guided short stems provide an anatomical curvature in the medial calcar region, thus, positioning is done individually alongside the calcar in the "round-the-corner" technique. Depending on the level of the neck's osteotomy, stems can be aligned individually in a large bandwidth of varus- and valgus anatomies. This differs from conventional total hip arthroplasty and potentially includes a severe learning curve. Given that a great variety of caput-collum-diaphyseal (CCD)-angles can be retained, the reconstruction of femoro-acetabular offsets can be achieved precisely. However, particularly extensive varus- and valgus positioning has raised concerns in regard to stability and bone remodeling. The purpose of the present manuscript is to showcase the implantation technique in calcar-guided short-stem THA and to summarize short-term clinical and radiological results.

This protocol describes the round-the-corner technique and the individualized stem-positioning of calcar-guided short stems alongside the medial calcar, depending on the level of the osteotomy. This differs from conventional total hip arthroplasty and includes a learning curve.

Bone- and soft-tissue sparing short stems are increasingly used in total hip arthroplasty (THA). However, there are a large variety of models of short ...

A Blood-based Test for the Detection of ROS1 and RET Fusion Transcripts from Circulating Ribonucleic Acid Using Digital Polymerase Chain Reaction

We have developed novel methods for the isolation and characterization of tumor-derived circulating ribonucleic acid (cRNA) for blood-based liquid biopsy. Robust detection of cRNA recovered from blood represents a solution to a critical unmet need in clinical diagnostics. The test begins with the collection of whole blood into blood collection tubes containing preservatives that stabilize cRNA. Cell-free, exosomal, and platelet-associated RNA is isolated from plasma in this test system. The cRNA is reverse transcribed to complementary DNA (cDNA) and amplified using digital polymerase chain reaction (dPCR). Samples are evaluated for both the target biomarker as well as a control gene. Test validation included limit of detection, accuracy, and robustness studies with analytic samples. The method developed as a result of these studies reproducibly detect multiple fusion variants for ROS1 (C-Ros proto-oncogene 1; 8 variants) and RET (rearranged during transfection proto-oncogene; 8 variants). The sample processing workflow has been optimized so that test results can consistently be generated within 72 hours of sample receipt.

Detection of circulating ribonucleic acid (cRNA) from blood is an unmet need in clinical diagnostics. Here we describe methods that characterize cRNA from non-small cell lung cancer patients using sensitive and specific digital polymerase chain reaction. The tests meet design requirements to detect fusion variants within 72 hours.

We have developed novel methods for the isolation and characterization of tumor-derived circulating ribonucleic acid (cRNA) for blood-based liquid biopsy. ...

Remote Laboratory Management: Respiratory Virus Diagnostics

An uptick in recent pandemics (Ebola, Zika, MERS, influenza, etc.) underlines the need for a more 'nimble,' coordinated response that addresses a multitude of issues ranging from transportation, access, facilities, equipment, and communication to provider training. To address this need, we have developed an innovative, scalable, logistics-enhanced, mobile, laboratory facility for emergencies and epidemics in resource-constrained global settings. Utilizing a background in clinical operations as an academic medical center, we designed a rapidly-deployable, modular BSL-2 and BSL-3 facility with user-friendly software for tracking and management of drugs and supplies in remote regions during epidemics and outbreaks. Here, we present our intermodal, mobile, expandable shipping-container laboratory units. The design of the laboratory facilitates off-grid usage by minimizing power consumption and allowing alternate water sources. The unit's information communication technology (ICT) platform provides (i) user-friendly tablet-based documentation, (ii) enhanced tracking of patients and supplies, and (iii) integrated communication onsite with built-in telehealth capabilities. To ensure quality in remote environments, we have developed a checklist for a basic laboratory workflow and a protocol for respiratory viral diagnosis using reverse-transcription polymerase chain reaction (RT-PCR). As described, this innovative and comprehensive approach allows for the provision of laboratory capability in resource-limited global environments.

A rapidly-deployable, off-grid laboratory has been designed and built for remote, resource-constrained global settings. The features and critical aspects of the logistically-enhanced, expandable, multifunctional laboratory modules are explored. A checklist for a basic laboratory workflow and a protocol for a respiratory viral diagnostic test are developed and presented.

An uptick in recent pandemics (Ebola, Zika, MERS, influenza, etc.) underlines the need for a more 'nimble,' coordinated response that addresses a ...

In Vivo Mouse Model of Spinal Implant Infection

Spine implant infections portend poor outcomes as diagnosis is challenging and surgical eradication is at odds with mechanical spinal stability. The purpose of this method is to describe a novel mouse model of spinal implant infection (SII) that was created to provide an inexpensive, rapid, and accurate in vivo tool to test potential therapeutics and treatment strategies for spinal implant infections.
In this method, we present a model of posterior-approach spinal surgery in which a stainless-steel k-wire is transfixed into the L4 spinous process of 12-week old C57BL/6J wild-type mice and inoculated with 1 x 103 CFU of a bioluminescent strain of Staphylococcus aureus Xen36 bacteria. Mice are then longitudinally imaged for bioluminescence in vivo on post-operative days 0, 1, 3, 5, 7, 10, 14, 18, 21, 25, 28, and 35. Bioluminescence imaging (BLI) signals from a standardized field of view are quantified to measure in vivo bacterial burden.
To quantify bacteria adhering to implants and peri-implant tissue, mice are euthanized and the implant and surrounding soft tissue are harvested. Bacteria are detached from the implant by sonication, cultured overnight and then colony forming units (CFUs) are counted. The results acquired from this method include longitudinal bacterial counts as measured by in vivo S. aureus bioluminescence (mean maximum flux) and CFU counts following euthanasia.
While prior animal models of instrumented spine infection have involved invasive, ex vivo tissue analysis, the mouse model of SII presented in this paper leverages noninvasive, real time in vivo optical imaging of bioluminescent bacteria to replace static tissue study. Applications of the model are broad and may include utilizing alternative bioluminescent bacterial strains, incorporating other types of genetically engineered mice to contemporaneously study host immune response, and evaluating current or investigating new diagnostic and therapeutic modalities such as antibiotics or implant coatings.

The protocol describes a novel in vivo mouse model of spinal implant infection where a stainless-steel k-wire implant is infected with bioluminescent Staphylococcus aureus Xen36. Bacterial burden is monitored longitudinally with bioluminescent imaging and confirmed with colony forming unit counts after euthanasia.

Spine implant infections portend poor outcomes as diagnosis is challenging and surgical eradication is at odds with mechanical spinal stability. The ...

Dynamic Navigation for Dental Implant Placement

In modern implantology, the application of surgical navigation systems is becoming increasingly important. In addition to static surgical navigation methods, a guide-independent dynamic navigation implant placement procedure is becoming more widespread. The procedure is based on computer-guided dental implant placement utilizing optical control. This work aims to demonstrate the technical steps of a new dynamic computer-aided implant surgery (DCAIS)&#160;system (design, calibration, surgery) and check the accuracy of the results. Based on cone-beam computed tomography (CBCT) scans, the exact positions of implants are determined with dedicated software. The first step of the operation is the calibration of the navigation system, which can be performed in two ways: 1) based on CBCT images taken with a marker or 2) based on CBCT images without markers. Implants are inserted with the aid of real-time navigation according to the preoperative plans. The accuracy of the interventions can be evaluated based on postoperative CBCT images. The preoperative images containing the planned positions of the implants and postoperative CBCT images were compared based on the angulation (degree), platform, and apical deviation (mm) of the implants. To evaluate the data, we calculated the standard deviation (SD), mean, and standard error of the mean (SEM) of deviations within planned and performed implant positions. Differences between the two calibration methods were compared based on this data. Based on the interventions performed so far, the use of DCAIS allows for high-precision implant placement. A calibration system that does not require labeled CBCT recording allows for surgical intervention with similar accuracy as a system that uses labeling. The accuracy of the intervention can be improved by training.

Dynamic computer-aided implant surgery (DCAIS) is a controlled implant surgical placement method performed without a surgical template using optical control. The real-time intraoperative control of movement and position of the surgical device simplifies the procedure and gives more freedom to the surgeon, providing similar precision as static navigation methods.

In modern implantology, the application of surgical navigation systems is becoming increasingly important. In addition to static surgical navigation ...

The Use of Mixed Reality in Custom-Made Revision Hip Arthroplasty: A First Case Report

The technology of 3D printing and visualization of anatomical structures is rapidly growing in various fields of medicine. A custom-made implant and mixed reality were used to perform complex revision hip arthroplasty in January 2019. The use of mixed reality allowed for a very good visualization of the structures and resulted in precise implant fixation. According to the authors' knowledge, this is the first described case report of the combined use of these two innovations. The diagnosis preceding the qualification for the procedure was the loosening of the left hip's acetabular component. Mixed reality headset and holograms prepared by engineers were used during the surgery. The operation was successful, and it was followed by early verticalization and patient rehabilitation. The team sees opportunities for technology development in joint arthroplasty, trauma, and orthopedic oncology.

A complex revision hip arthroplasty was performed using a custom-made implant and mixed reality technology. According to the authors&#39; knowledge, this is the first report of such procedure described in the literature.

The technology of 3D printing and visualization of anatomical structures is rapidly growing in various fields of medicine. A custom-made implant and mixed ...

Measuring Single-Cell Mitochondrial DNA Copy Number and Heteroplasmy Using Digital Droplet Polymerase Chain Reaction

The mammalian mitochondrial (mt)DNA is a small, circular, double-stranded, intra-mitochondrial DNA molecule, encoding 13 subunits of the electron transport chain. Unlike the diploid nuclear genome, most cells contain many more copies of mtDNA, ranging from less than 100 to over 200,000 copies depending on cell type. MtDNA copy number is increasingly used as a biomarker for a number of age-related degenerative conditions and diseases, and thus, accurate measurement of the mtDNA copy number is becoming a key tool in both research and diagnostic settings. Mutations in the mtDNA, often occurring as single nucleotide polymorphisms (SNPs) or deletions, can either exist in all copies of the mtDNA within the cell (termed homoplasmy) or as a mixture of mutated and WT mtDNA copies (termed heteroplasmy). Heteroplasmic mtDNA mutations are a major cause of clinical mitochondrial pathology, either in rare diseases or in a growing number of common late-onset diseases such as Parkinson's disease. Determining the level of heteroplasmy present in cells is a critical step in the diagnosis of rare mitochondrial diseases and in research aimed at understanding common late-onset disorders where mitochondria may play a role. MtDNA copy number and heteroplasmy have traditionally been measured by quantitative (q)PCR-based assays or deep sequencing. However, the recent introduction of ddPCR technology has provided an alternative method for measuring both parameters. It offers several advantages over existing methods, including the ability to measure absolute mtDNA copy number and sufficient sensitivity to make accurate measurements from single cells even at low copy numbers. Presented here is a detailed protocol describing the measurement of mtDNA copy number in single cells using ddPCR, referred to as droplet generation PCR henceforth, with the option for simultaneous measurement of heteroplasmy in cells with mtDNA deletions. The possibility of expanding this method to measure heteroplasmy in cells with mtDNA SNPs is also discussed.

Here we present a protocol for measuring absolute mitochondrial (mt)DNA copy number and mtDNA deletion heteroplasmy levels in single cells.