Aim. The aim of this study was to develop an in-house multiplex PCR real-time assay on the LightCycler 480 system (Roche, Basel, Switzerland) with the aim of rapid detection of common pathogens in prosthetic joint infections (PJI), followed by validation on clinical samples (sonication fluid and tissue biopsies) routinely collected for PJI diagnosis. Methods. Using the PrimerQuest and CLC WorkBench tool, we designed six primer sets with specific fluorescently labelled TaqMan probes for the nuc gene in different Staphylococcus species (S. aureus, S. epidermidis, S. capitis, S. lugdunensis, S. hominis, S. haemolyticus). In addition, primers previously developed by Renz et al. (2022) for C. acnes were integrated into our assay with internal control of isolation, leading to the development of specific mPCR assay with seven included targets. Analytical sensitivity and specificity were evaluated using reference bacterial strains. To determine the assay's limit of detection (LOD), we conducted serial dilutions of eluates containing known concentrations of bacterial DNA copies/µl. The overall LOD in spiked clinical samples, including sample preparation and DNA isolation on MagnaPure24, was measured through 10-fold serial dilutions (from 10. 9. to 10. -1. CFU/ml) including additional dilutions of 5000, 500, 50 and 5 CFU/ml. Results. The results with LOD in serial dilutions of eluates and spiked clinical samples, together with analytical sensitivity and specificity, are shown in Table 1. Conclusion. The mPCR assay showed excellent analytical sensitivity and specificity, but with considerably lower LOD after sample preparation and further DNA isolation in spiked clinical samples. Although still promising in diagnostics of acute infections, the use of mPCR could be challenging in chronic, low-grade infections with lower microbial burden. Nevertheless, PCR offers significant advantages in terms of speed and can shorten the time to result, especially for C. acnes infections. Additionally, it represents a promising complementary approach in patients with suspected PJI on antibiotic therapy with negative culture results. For any tables or figures, please contact the authors directly

Aim. Currently, despite a thorough diagnostic work up, around ten percent of the presumed aseptic revisions turn out to have unexpected positive cultures during the revision procedure. The purpose of this study was to evaluate the negative predictive value (ruling out) of the automated multiplex PCR Unyvero i60 implant and tissue infection (ITI) cartridge (U-ITI) system for the detection of microorganisms in synovial fluid obtained intraoperatively. Methods. A prospective study was conducted with 200 patients undergoing a one-stage knee or hip revision. In all patients six intraoperative tissue cultures were taken and a sample of synovial fluid which was analyzed as a culture and with the multiplex PCR U-ITI system. The primary outcome measure was the negative predictive value (NPV) of the multiplex PCR U-ITI system compared to the intraoperative tissue cultures to reliable rule out an infection. Results. The NPV of the multiplex PCR U-ITI system of synovial fluid compared to tissue cultures in knee and hip revisions was 96.8% and 92.5%, respectively. In addition, cultures require several days for growth whereas the automated mPCR U-ITI system provides results within five hours. Conclusions. The multiplex PCR U-ITI system is a quick additional test to conventional cultures in presumed aseptic knee and hip revisions for reliable ruling out of an underlying infective cause. With this simple test antibiotic overtreatment as well as undertreatment after one-stage revision arthroplasty can be avoided which can directly result in a reduction in length of hospital stay, hospital costs and possible antibiotic resistance development

In this study we evaluated the performance of the newly available ITI-Cartridge (UniveroTM i60 implant and tissue infection (ITI) multiplex polymerase chain reaction (PCR) System, Curetis®, Holzgerlingen, Germany) in diagnosing periprosthetic joint infection (PJI). 30 patients that received an operative revision in the orthopaedic department of the University Hospital Bonn due to suspected PJI or aseptic loosening of a painful total hip or knee arthroplasty between Januar 2014 and November 2014 were included in this retrospective study. The microbiological workup included a minimum of three periprosthetic tissue specimens, joint aspirate and the explanted foreign body for sonication were investigated. Additionally, histopathological examination of the periprosthetic membranes, cell counting of the joint aspirate and multiplex PCR diagnostic of the sonication fluid cultures and of the joint aspirate were performed. All patients were summarized in two diffrent groups (PJI vs. free of infection) according to the classification of the International Consensus Group on Periprosthetic Joint Infection [4]. In our collective sonication fluid cultures had a sensitivity of 88.89% with a specificity of 61.54%. Other microbiological specimens, especially tissue samples and joint aspirates showed both a sensitivity of 66.67%, and a specificity of 92.31% and respectively 84.62%. PCR-based rapid testing of sonication fluid yielded out a sensitivity of 50% with a specificity of 100%. PCR of the joint aspirate documented a slightly better sensitivity of 55.56 % with a specificity of 100%. When summarized these two PCRs the sensitivity rose to 66.67% with a specificity of 100%. In summary, PCR-diagnostic is an additional method to gain ancillary informations in diagnosing PJI but it has to be interpretated carefully in synopsis with the results obtained from tissue cultures, sonication fluid cultures, histopathological examination and clinical course. The performance of the newly available multiplex PCR system ITI-Cartridge did not persuade us, so that PCR diagnostic of sonication fluid culture or joint aspirate was not included in our algorythm of diagnosing PJI

Aim. When treating periprosthetic joint infections with a two-stage procedure, antibiotic-impregnated spacers are used in the interval between removal of prosthesis and reimplantation. The spacer provides local antibiotics; however, it may also act as foreign-body that can be colonized by microorganisms. According to our experience, cultures of sonicated spacers are most often negative. The objective of our study was to investigate whether PCR analysis would improve the detection of bacteria in the spacer sonication fluid. Method. A prospective monocentric study was performed at Lausanne University Hospital from September 2014 until January 2016. Inclusion criteria were two-stage procedure for prosthetic infection and agreement of the patient to participate in the study. For a two-stage procedure the interval before reimplantation ranged between 2 and 8 weeks. Spacers were made of cement impregnated with gentamycin, tobramycin and vancomycin. Cultures of intraoperative deep tissues samples from first and second stage procedures, prosthesis sonication and spacer sonication were analyzed. Multiplex-PCR. *. , pan-bacterial PCR (16S), and a Staphylococcus-specific PCR analysis were performed on the sonicated spacer fluid. Results. 23 patients were identified (12 hip, 10 knee and 1 ankle replacements). Initial infection was caused by Staphylococcus aureus (27%), Streptococcus epidermidis (27%), S. dysgalactiae (13%), S. milleri (9%), S. pneumoniae (4%), S. capitis (4%), S. salivarus (4%), P. acnes (4%), E. faecalis (4%) and C. fetus (4%). At reimplantation, cultures of tissue samples and spacer sonication fluid were all negative. Of culture-negative samples, the PCR analyses were negative except for 5 cases. 4 cases of infection recurrence were observed, with bacteria different than for the initial infection in 3 cases. For these cases, no germs were detected in the spacer sonication fluid by neither cultures nor PCR. Conclusions. The 3 different PCR analyses performed did not detect any bacteria in spacer sonication fluid that was culture-negative. In our study, PCR did not improve the bacterial detection and did not help to predict whether the patient will present a recurrence of infection. Prosthetic 2-stage exchange with short interval and antibiotic-impregnated spacer is an efficient treatment to eradicate infection as both culture- and molecular-based methods were unable to detect bacteria in spacer sonication fluid after reimplantation

Aim. Bone and Joint Infections (BJIs) present with non-specific symptoms and can be caused by a wide variety of bacteria and fungi, including many anaerobes and microorganisms that can be challenging to culture or identify by traditional microbiological methods. Clinicians currently rely primarily on culture to identify the pathogen(s) responsible for infection. The BioFire. ®. FilmArray. ®. Bone and Joint Infection (BJI) Panel (BioFire Diagnostics, Salt Lake City, UT) was designed to detect 15 gram-positive (seven anaerobes), 14 gram-negative bacteria (one anaerobe), two yeast, and eight antimicrobial resistance (AMR) genes from synovial fluid specimens in an hour. The objective of this study was to evaluate the performance of an Investigational Use Only (IUO) version of the BioFire BJI Panel (BBJIP) compared to conventional used as reference methods. Method. In a monocentric study, leftover synovial fluid specimens were collected in a single institution including 4 hospitals and tested using conventional bacterial culture (Standard of Care (SoC)) according to routine procedures following French national recommendations. Specimen has been placed in a refrigerator (4°C) as soon as possible after collection and stored for less than or equal to 7 days before enrollment. Performance of the IUO version of the BBJIP was determined by comparison to SoC for species identification. Results. To date, 201 specimens have been collected and tested using BBJIP. A total of 39 pathogens were obtained in culture. Compared to SoC culture, the overall PPA was 89.7% (35 TP, 4 FN (SA, 1; Strepto Spp, 2; P. micra, 1) and the overall NPA was 99.7% with 16 FP for a total of 5374 bacterial targets screened. Two complementary molecular tests using home-made PCR are underway to definitively conclude about the FN et FP for BBJIP observed in the preset study. Conclusions. The BioFire BJI Panel appears as a promising, sensitive, specific, and robust test for rapid detection of 31 microorganisms (including anaerobes) and eight AMR genes in synovial fluid specimens. The number of pathogens and resistance markers included in the BioFire BJI Panel, together with a reduced time-to-result and increased diagnostic yield compared to culture, is expected to aid in the management of BJIs

Aim. Periprosthetic joint infection (PJI) is nowadays the most important problem leading to failure in primary and revision total knee (TKA) and total hip arthroplasty (THA), therefore accurate diagnosis of PJI is necessary. We evaluated a commercial multiplex PCR system1 for diagnosis of PJI in joint aspiration fluids prior to surgery. Method. A total of 32 patients were included in the study. Twenty-four patients had TKA and eight had THA. Joint aspiration fluids were examined by standard bacteriological procedures. Excess material of joint aspirates was frozen at −20°C until testing by multiplex PCR1. Inclusion criteria were a minimum leucocyte count of 2.000 per ml and at least 60% of polymorphonucleaur neutrophils (PNN) in the joint aspiration fluid. Results. For 21 patients with TKA, both standard bacteriological culture and PCR1 were negative. In these patients the mean leucocyte count in the joint fluid was 15.385/ml with 80% PNN. For three patients culture was negative, but PCR1 was positive. In one patient PCR1 detected Corynebacterium sp. which was considered as contamination as this patient had crystal arthropathy; for the second patient Propionibacterium acnes was detected by PCR1, this patient was treated as having an infection of unknown origin in another hospital. For the third patient PCR1 detected Pseudomonas aeruginosa. This patient was known as having chronic P. aeruginosa infection of his TKA and joint aspiration was done shortly after arrest of antibiotic therapy by ciprofloxacin. The mean leucocyte count in the patients with positive PCR was 61.800/ml with 89% PNN. In three of the eight patients with THA, standard bacterial culture and PCR1 were both negative. The mean leucocyte count in joint aspirates of these patients was 10.087/ml with 77% PNN. In five patients with THA, both culture and PCR1 were positive and concordant. In one case culture and PCR1 detected Staphylococcus aureus, and in the other culture and PCR1 detected P. acnes. In two cases culture grew S. epidermidis and PCR1 detected coagulase negative Staphylococcus. In the fifth patient culture grew C. jeikeium and PCR1 detected Corynebacterium spp. Conclusions. We found concordant results for culture and PCR1 in all eight patients with THA and in 22/24 patients (92%) with TKA. Multiplex PCR1 results are available in 4 hours whereas culture results may demand several days. The commercial multiplex PCR system1 designed for diagnosis of implant and tissue infection can be helpful for the diagnosis of PJI. *Unyvero i60©, Curetis Strasbourg, France

Aim

Prompt recognition and identification of the causative microorganism in acute septic arthritis of native and prosthetic joints is vital to increase the chances of successful treatment. The aim of this study was to independently assess the diagnostic accuracy of the multiplex BIOFIRE® Joint Infection (JI) Panel (investigational use only) in synovial fluid for rapid diagnosis

Background:

GeneXpert, a new, rapid molecular diagnostic test is recommended as the first line investigation for suspected pulmonary TB in areas of high HIV prevalence or drug resistance, yet it has not been validated for the diagnosis of musculoskeletal TB.

Aim

To determine whether rep-PCR genotyping can improve the diagnosis of coagulase-negative staphylococci(CoNS)bone and joint infection relative to the standard method based on phenotypic identification.

Aim. We prospectively evaluated four different microbiological tools for diagnostics of prosthetic joint infections (PJI), and assessed their impact on the categorization of infection according to EBJIS guidelines. We compared culture, in-house real-time mPCR for S. aureus, S. lugdunensis, S. hominis, S. epidermidis, S. capitis, S. haemolyticus, C. acnes (mPCR), broad-spectrum PCR (Molzym) with 16S rRNA V3-V4 amplicon Sanger sequencing (16S PCR), and 16S rRNA V3-V4 amplicon next-generation sequencing (16S NGS) on MiSeq (Ilumina). Methods. A total of 341 samples (sonication fluid, tissue biopsy, synovial fluid) were collected from 32 patients with suspected PJI who underwent 56 revision surgeries at the Orthopaedic Centre University Hospital Ljubljana, between 2022 and 2024. Samples were processed using standard protocols for routine culture, followed by DNA isolation using the MagnaPure24 (Roche). All samples were tested with mPCR, and an additional ≥4 samples from each revision (244 in total) were subjected to further metagenomic analysis. Culture results were considered positive if the same microorganism was detected in ≥2 samples, ≥50 CFU/ml were present in the sonication fluid, or ≥1 sample was positive for a more virulent microorganism or if the patient had received antibiotic treatment. Results. Each tool demonstrated high sensitivity for correct EBJIS categorization (100% culture and 16S NGS, 96.88% mPCR and 16S PCR). The highest specificity was observed with mPCR and 16S PCR (87.5%), while culture (79.17%) and NGS (37.5%) showed lower specificity. In 27% (15/56) of revisions, all microbiological tests were negative, although infection was confirmed with histology in one case, and four cases were classified as infection-likely based on clinical signs. In 20% (11/56) of cases, all microbiological tests were positive; in three cases a combination of other EBJIS criteria (without microbiology) categorized the episodes as infection-likely and one as infection-unlikely, emphasizing the importance of microbiological tests in diagnostic criteria. In 43% (24/56) of revisions categorized as infection-unlikely using a combination of other EBJIS criteria, five had positive culture, and three had positive mPCR and 16S PCR. Fifteen (62%) had positive 16S NGS, 12 due to a low number of reads, which may indicate low-grade infection or possible contamination. Conclusion. To date, no test can be established as the ultimate gold standard. The lack of interpretation criteria can result in low specificity of some methods, as the threshold is difficult to determine. A multidisciplinary approach with combination of microbiological tools is still considered the most efficient

Aim. Despite the availability of numerous tests, the diagnosis of periprosthetic infection (PJI) continues to be complex. Although several studies have suggested that coagulation-related markers, such as D-dimer and fibrinogen, may be promising tools in the diagnosis of prosthetic infections, their role is still controversial. The aim of this study is to evaluate the diagnostic accuracy of serum D-dimer and fibrinogen in patients with painful total knee replacement. Method. 83 patients with painful total knee replacement and suspected peri-prosthetic infection were included. All patients underwent pre-operative blood tests to evaluate inflammation indices (ESR and CRP) and serum D-Dimer and Fibrinogen levels. The diagnostic performance of the tests was assessed using the ICM definition as the gold standard. The diagnostic accuracy of the D-dimer and fibrinogen was measured by assessing sensitivity, specificity and by calculating the area under the ROC curve. Results. The definition of prosthetic infection based on the ICM criteria has made it possible to classify 40 peri-prosthetic infections and 43 aseptic failures. The mean value of fibrinogen, D-Dimer, VES and PCR observed in patients with prosthetic infection was significantly higher than in patients with aseptic failure [fibrinogen 468 mg / dl vs 331 mg / dl, p <0.001; D-Dimero 2177 ng/mL vs. 875 ng / mL, p <0.005], ESR 49 mm / hr vs 24 mm/h, p <0.001; PCR 25.5 mg /L vs 8.9 mg/L, p <0.001]. The optimal threshold value of the fibrinogen indicative of the presence of infection was 418 mg/dl, with a sensitivity of 72% and a specificity of 88%. The serum concentration of d-dimer greater than 945 ng / ml showed a sensitivity of 72.5% and a specificity of 76.7%. Conclusions. Although in this multicenter prospective study we found that serum D-dimer may have significantly higher statistical values in PJI than aseptic failures, its diagnostic power appears however limited when compared with other markers including plasma fibrinogen. Fibrinogen is regularly analyzed before surgery, the evaluation of this marker does not involve additional costs. The diagnostic accuracy appears to be similar to that of classic markers such as the level of PCR and VES. Plasma D-dimer may have a limited value in the diagnosis of PJI unlike plasma fibrinogen which has shown moderate sensitivity and excellent specificity. However, in our limited series of cases, both tests cannot be used alone in the diagnosis of infection but could contribute to the diagnosis if contextualized to ves and pcr

Introduction. Many fluoroscopic studies on total knee arthroplasty (TKA) have identified kinematic variabilities compared to the normal knee, with many subjects experiencing paradoxical motion patterns. The intent of this study was to investigate the results of a newly designed PCR TKA to determine kinematic variabilities and assess these kinematic patterns with those previously documented for the normal knee. Methods. The study involves determining the in vivo kinematics for 80 subjects compared to the normal knee. 10 subjects have a normal knee, 40 have a Journey II PCR TKA and 40 subjects with the Journey II XR TKA (BCR). Although all PCR subjects have been evaluated, we are continuing to evaluate subjects with a BCR TKA. All TKAs were performed by a single surgeon and deemed clinically successful. All subjects performed a deep knee bend from full extension to maximum flexion while under fluoroscopic surveillance. Kinematics were calculated via 3D-to-2D registration at 30° increments from full extension to maximum flexion. Anterior/posterior translation of the medial (MAP) and lateral (LAP) femoral condyles and femorotibial axial rotation were compared during ranges of motion in relation to the function of the cruciate ligaments. Results. Of the 40 PCR TKAs, the average overall flexion was 112.6°, while the average for normal subjects was 139.0°. Initial BCR subjects revealed a higher than expected 128.0°. From 0=30° knee flexion, PCR subjects demonstrated −4.74±4.94 mm of posterior LAP movement, −2.04±4.07 mm of MAP movement and 3.61±8.13° of external axial rotation. In the same range of motion, normal subjects exhibited −8.80±3.32 mm of LAP movement, −3.81±1.03 mm of MAP movement and an axial rotation of 11.34±3.78°. From 30=90° knee flexion, PCR subjects demonstrated 4.37±8.26 mm of LAP movement, 0.12±7.95 mm of MAP movement and 0.79±11.43° of axial rotation. In the same range of motion, normal subjects exhibited −4.28±3.13 mm of LAP movement, −1.11±2.76 mm of MAP movement and axial rotation of 6.54±4.33°. From 0°-maximum flexion, PCR subjects demonstrated −2.71±5.37 mm of LAP movement, 1.79±4.88 mm of MAP movement and 5.99±5.26° of axial rotation. In the same range of motion, normal subjects exhibited −17.83±6.04 mm of LAP movement, −9.11±4.93 mm of MAP movement and axial rotation of 23.66±7.81°. Overall, the BCR subject displayed kinematic patterns similar to those of a normal knee; more detailed numbers will be presented in the presentation. Discussion. Subjects having a PCR TKA experienced excellent weight-bearing flexion and kinematic patterns similar to the normal knee, but less in magnitude. These subjects experienced posterior femoral rollback in early and late flexion. During mid-flexion, subjects having a PCR TKA did experience some variable motion patterns, which may be due to the absence of the ACL. Subjects having a BCR TKA experienced more continuous rollback throughout flexion, more similar to the normal knee. Similar to the normal knee, subjects having a PCR TKA did experience progressive axial rotation throughout knee flexion (Figures). Significance. While they still experience normal-like rollback during early (0°–30°) and late flexion (90°-120°), subjects with a PCR TKA consistently demonstrated Anteriorization of the joint in mid-flexion

Aim. Diagnosis of periprosthetic shoulder infections (PSI) is difficult as they are mostly caused by low-virulent bacteria and patients do not show typical infection signs, such as elevated blood markers, wound leakage, or red and swollen skin. Ultrasound-guided biopsies for culture may therefore be an alternative for mini-open biopsies as less costly and invasive method. The aim of this study was to determine the diagnostic value and reliability of ultrasound-guided biopsies for cultures alone and in combination polymerase chain reaction (PCR), and/or synovial markers for preoperative diagnosis of PSI in patients undergoing revision shoulder surgery. Method. A prospective explorative diagnostic cohort study was performed including patients undergoing revision shoulder replacement surgery. A shoulder puncture was taken preoperatively before incision to collect synovial fluid for interleukin-6 (IL-6), calprotectin, WBC, polymorphonuclear cells determination. Prior to revision surgery, six ultrasound-guided synovial tissue biopsies were collected for culture and two additional for PCR analysis. Six routine care tissue biopsies were taken during revision surgery and served as reference standard. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV; primary outcome measure), and accuracy were calculated for ultrasound-guided biopsies, and synovial markers, and combinations of these. Results. Fifty-five patients were included. In 24 patients, routine tissue cultures were positive for infection. Cultures from ultrasound-guided biopsies diagnosed an infection in 7 of these patients, yielding a sensitivity, specificity, PPV, NPV, and accuracy of 29.2%, 93.5%, 77.8%, 63.0%, and 65.6%, respectively. Ultrasound-guided biopsies in combination with synovial WBC increased the NPV to 76.7% and accuracy to 73.8%. When synovial WBC and calprotectin were combined with ultrasound-guided biopsies, it resulted in a better diagnostic value: sensitivity 69.2%, specificity 80.0%, PPV 69.2%, NPV 80.0%, and accuracy 75.8%. Ultrasound-guided biopsies in combination with calprotectin and ESR yielded a sensitivity of 50.0%, specificity of 93.8%, PPV of 80.0%, NPV of 78.9%, and accuracy of 79.2%. Synovial fluid was obtained in 42 patients. Sensitivities of WBC, PMN, IL-6, and calprotectin were between 25.0% and 35.7%, specificities between 89.5% and 95.0%, PPVs between 60.0% and 83.3%, NPVs between 65.4% and 69.4%, and accuracies between 64.5% and 70.6%. Conclusions. In this prospective study we showed that ultrasound-guided biopsies for cultures alone and in combination with PCR and/or synovial markers are not reliable enough to use in clinical practice for the preoperative diagnosis of low grade PSI

Background. While posterior cruciate retaining (PCR) implants are a more common total knee arthroplasty (TKA) design, newer bi-cruciate retaining (BCR) TKAs are now being considered as an option for many patients, especially those that are younger. While PCR TKAs remove the ACL, the BCR TKA designs keep both cruciate ligaments intact, as it is believed that the resection of the ACL greatly affects the overall kinematic patterns of TKA designs. Various fluoroscopic studies have focused on determination of kinematics but haven't defined differentiators that affect motion patterns. This research study assesses the importance of the cruciate ligaments and femoral geometry for Bi-Cruciate Retaining (BCR) and Posterior Cruciate Retaining (PCR) TKAs having the same femoral component, compared to the normal knee. Methods. The in vivo 3D kinematics were determined for 40 subjects having a PCR TKA, 10 having a BCR TKA, and 10 having a normal knee, in a retrospective study. All TKA subjects had the same femoral component. All subjects performed a deep knee bend under fluoroscopic surveillance. The kinematics were determined during early flexion (ACL dominant), mid flexion (ACL/PCL transition) and deep flexion (PCL dominant). Results. During the first 30 degrees of flexion, the ACL played an important role, as subjects having a BCR TKA experienced kinematic patterns more similar to the normal knee. During mid flexion, both TKAs experienced random kinematic patterns, which could be due to the ACL and PCL being less active or resected in PCR TKA. In deeper flexion, both TKAs experienced kinematic patterns similar to the normal knee, thus supporting the assumption that the PCL played a dominant role [Fig. 1, Fig. 2]. All three groups generally experienced progressive axial rotation throughout flexion [Fig. 3]. On average, subjects having a PCR TKA experienced 112.3° of flexion, which was greater than the BCR subjects. Conclusions. Both the BCR TKA and normal groups experienced similar kinematic patterns, but the femoral geometrical differences from the anatomical condition may have influenced decreased motion compared to the normal knee. Both TKAs experienced similar kinematic patterns in deeper flexion, with the PCR TKA experiencing excellent weight-bearing flexion. Results from this study suggest that the cruciate ligaments can play a role in kinematics, but femoral geometry working with the ligaments may be an option to consider

Aims. Microbiological diagnosis of bone and joint infections (BJIs) is pivotal. However, no consensus exists about the best choice for techniques to be used and the best indications for molecular methods. Our objectives were: (i) to compare the performance of various microbiological diagnostic methods (cultural and molecular) on synovial fluid specimens and (ii) to select an algorithm for optimizing the diagnosis of BJIs in adults. Methods. This prospective multicentric study (in Lyon and Saint-Etienne, France) included 423 joint fluid samples, collected from 333 adult patients (median age 69 years) suspected for BJI on the basis of medical history and clinical symptoms. For each inclusion, joint fluid and blood culture were collected concomitantly. The synovial fluid was also inoculated into blood culture bottles. Cytology, culture (using 5 solid media and an enrichment broth, incubated for 15 days), universal 16S rRNA PCR and PCR targeting Staphylococcus spp, S.aureus, Streptococcus spp, S.pneumoniae, Kingella kingae, Borrelia burgdorferi and Propionibacterium acnes were systematically performed on synovial fluid. Results. Prosthetic materials were present in 65.0% of the cases and 31.7% of the patients had received antibiotics in the 15 days before puncture. Out of 423 joint fluids, 265 (62.6%) were positive by at least one diagnostic technique (cultural or molecular): 219 mono- and 46 poly-microbial, for a total of 322 bacteria. Identified bacteria were staphylococci in 54.0%, streptococci-enterococci in 15.2%, Gram-negative bacilli in 14.0%, anaerobic species in 10.9% and other bacteria in 5.9% of cases. Comparing the individual performance of each cultural technique, blood culture bottles showed the highest rate of positivity (detecting 61.4 and 58.4% of the bacteria, for the paediatric and anaerobic bottles, respectively) but cannot be performed alone and require to be combined with solid media. The 16S rDNA PCR was positive in only 49.2% of the cases whereas higher detection was obtained with specific PCR. Blood cultures performed concomitantly with joint puncture were positive in only 9.7% of the cases. Conclusions. In order to simplify the culture procedures and to precise the place of PCR for synovial fluid, we propose the following algorithm: joint fluids should be inoculated onto 3 solid media (blood and chocolate agars for 2 days, anaerobic blood agar for 10 days), associated with inoculation into blood culture bottles for 10 days. If culture remains negative, 16S rDNA PCR and/or Staphylococcus PCR should be added. Applying this algorithm on our cohort, 93.6% of the bacteria would have been detected

3D printing and Bioprinting technologies are becoming increasingly popular in surgery to provide a solution for the regeneration of healthy tissues. The aim of our project is the regeneration of articular cartilage via bioprinting means, to manage isolated chondral defects. Chrondrogenic hydrogel (chondrogel: GelMa + TGF-b3 and BMP6) was prepared and sterilised in our lab following our standard protocols. Human adipose-derived mesenchymal stem cells were harvested from the infrapatellar fat pad of patients undergoing total knee joint replacements and incorporated in the hydrogel according to our published protocols. The chondrogenic properties of the chondrogel have been tested (histology, immunohistochemistry, PCR, immunofluorescence, gene analysis and 2. nd. harmonic generation microscopy) in vitro and in an ex-vivo model of human articular defect and compared with standard culture systems where the growth factors are added to the media at repeated intervals. The in-vitro analysis showed that the formation of hyaline cartilage pellet was comparable between the two strategies, with a similar metabolic activity of the cells. These results have been confirmed in the ex-vivo model: hyaline-like cartilage was observed within the chondral defect in both the chondrogel group and the control group after 28 days in culture. The use of bioprinting techniques in vivo requires the ability of stem cells to access growth factors directly in the environment they are in, as opposed to in vitro techniques where these factors are provided externally at recurrent intervals. This study showed the successful strategy of incorporating chondrogenic growth factors for the formation of hyaline-like cartilage in vitro and in an ex-vivo model of chondral loss. The incorporation of chondrogenic growth factors in a hydrogel is a possible strategy for articular cartilage regeneration

Specific and rapid detection methods for spinal tuberculosis, with sufficient sensitivity in HIV-1 co-infected individuals, are needed, to ensure early initiation of appropriate treatment to prevent physical disability and neurological fallout. In addition, understanding the systemic and local pathophysiology of spinal tuberculosis, and its interaction with HIV-1 infection, is crucial to guide future therapeutic interventions. We prospectively enrolled adult patients presenting with signs and symptoms of suspected spinal tuberculosis, at Groote Schuur Hospital, between November 2020 and December 2021. TB diagnostic testing was performed on open and CT-guided spinal biopsies using Xpert MTB/RIF Ultra compared to gold standards TB culture and histology. A highly sensitive droplet digital PCR assay for detecting and quantifying Mycobacterium tuberculosis complex (MTBC) and HIV-1 DNA was tested. Plasma inflammatory proteins were measured to assess systemic inflammation. Xpert Ultra had a high sensitivity of 94.7% and specificity of 100% for STB against TB culture and histology in both open and CT-guided biopsy samples. The ddPCR assay confirmed TB detection in 94% of patients with positive Xpert Ultra results. Four patients with negative TB diagnostic results had MTBC DNA detected by ddPCR. HIV-1 DNA was detected in the spinal tissues from all HIV-1-infected patients. MTBC DNA levels were significantly higher in HIV-1-co-infected spinal tissue samples (p< 0.01). We identified four biomarkers significantly associated with higher bacterial burden at the disease site (p< 0.01). Xpert Ultra and MTBC ddPCR improve the detection of STB. DdPCR can be utilized as an additional, highly sensitive tool for detecting and quantifying Mtb, in pathological samples that may be paucibacillary. These findings provide novel diagnostic and pathophysiologic insight into STB, in the context of HIV-1 infection, and provide rationale to include these tests in hospital and research settings for patients from communities burdened by TB and HIV-1

Aim. While 16S rRNA PCR - Sanger sequencing has paved the way for the diagnosis of culture-negative bacterial infections, it does not provide the composition of polymicrobial infections. We aimed to evaluate the performance of the Nanopore-based 16S rRNA metagenomic approach using partial-length amplification of the gene, and to explore its feasibility and suitability as a routine diagnostic tool for bone and joint infections (BJI) in a clinical laboratory. Method. Sixty-two clinical samples from patients with BJI were sequenced on MinION* using the in-house partial amplification of the 16S rRNA gene. BJI were defined based on the ICM Philly 2018 and EBJIS 2021 criteria. Among the 62 samples, 16 (26%) were culture-positive, including 6 polymicrobial infections, and 46 (74%) were culture-negative from mono- and polymicrobial infections based on Sanger-sequencing. Contamination, background noise definition, bacterial identification, and time-effectiveness issues were addressed. Results. Results were obtained within one day. Setting a threshold at 1% of total reads overcame the background noise issue and eased interpretation of clinical samples. The partial 16S rRNA metagenomics approach had a greater sensitivity compared both to the culture method and the Sanger sequencing. All the 16 culture-positive samples were confirmed with the metagenomic sequencing. Bacterial DNA was detected in 32 culture-negative samples (70%), with pathogens consistent with BJI. The 14 Nanopore negative samples included 7 negative results confirmed after implementation of other molecular techniques and 7 false-negative MinION results: 3 Kingella kingae infections detected after targeted-PCR only, 2 Staphylococcus aureus infections and 2 Pseudomonas aeruginosa infections sterile on agar plate media and detected only after implementation of blood culture media, advocating for the very low inoculum. Conclusions. The results discriminated polymicrobial samples, and gave accurate bacterial identifications compared to Sanger-based results. They confirmed that Nanopore technology is user-friendly as well as cost- and time-effective. They also indicated that 16S rRNA targeted metagenomics is a suitable approach to be implemented for routine diagnosis of culture-negative samples in clinical laboratories. * Oxford Nanopore Technologies

Introduction. Bicruciate-retaining (BiCR) total knee replacements (TKRs) were designed to improve implant performance; however, functional advantages during daily activity have yet to be demonstrated. Although level walking is a common way to analyze functionality, it has been shown to be a weak test for identifying gait abnormalities related to ACL pathologies. The goal of this study is to set up a functional motion analysis test that will examine the effects of the ACL in TKR patients by comparing knee kinematics, kinetics, and muscle activation patterns during level and downhill walking for patients with posterior-cruciate retaining (PCR) and BiCR TKRs. Methods. Motion and electromyography (EMG) data were collected simultaneously for 12 subjects (4/8 m/f, 64±11 years, 31.3±7.3 BMI, 6/6 right/left) with BiCR TKRs and 15 subjects (6/9 m/f, 67±7 years, 30.5±5.1 BMI, 4/11 right/left) with PCR TKRs during level and downhill walking using the point cluster marker set. Surface electrodes were placed on the vastus medialis obliquus (VMO), rectus femoris (RF), biceps femoris (BF), and semitendinosus (ST) muscles. EMG data are reported as percent relative voluntary contraction (%RVC), normalizing the signal during downhill walking to the mean maximum EMG value during level walking. Results. For level walking, there were no significant differences between groups in knee kinematics, kinetics, and EMG patterns. During downhill walking, subjects with BiCR implants showed significantly lower peak muscle activity in the VMO (73.9 ± 49.1%RVC for BiCR vs. 113 ± 24.0%RVC for PCR; p=0.045) and RF (96.0 ± 25.7%RVC for BiCR vs. 128 ± 28.6%RVC for PCR; p=0.018). There was also a trending higher knee peak flexion moment for the BiCR subjects (2.0 ± 0.6% BW*HT vs. 1.5 ± 0.6% BW*HT, p = .076), as well as significantly more knee flexion at heel strike (5.1 ± 4.7 degrees vs. 1.8 ± 2.8 degrees, p = 0.044) compared with the PCR group. Discussion. Retention of the ACL led to altered muscle recruitment during downhill walking in BiCR subjects compared with PCR subjects. In BiCR subjects, quadriceps activity was reduced during downhill walking compared to level walking. PCR subjects on average did not show this reduction, possibly in compensation for decreased knee stability. While there were only a few significant kinematic/kinetic differences, it appears that BiCR TKRs may offer some neuromuscular benefits during more strenuous tasks like downhill walking. In conclusion, level and downhill walking knee kinematics and kinetics together with the corresponding quadriceps and hamstrings EMG signals begin to build an overall picture of implant functionality during motion analysis testing

Aim. Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) is commonly associated with serious cases of community-onset skin and musculoskeletal infections (Co-SMSI). Molecular epidemiology analysis of CA-MRSA recovered from skin and soft tissues specimens is lacking in Latin America. This study aimed to identify phenotypic and genotypic features of MRSA isolates recovered from patients presenting Co-SMSI. Methods. Consecutive MRSA isolates recovered from Co-SMSI of patients admitted from March 2022 to January 2023 in a Brazilian teaching hospital were tested for antimicrobial resistance and characterized by their genotypic features. Identification was carried out by automated method and through MALDI-TOF MS. Antimicrobial susceptibility was tested by disk diffusion, broth microdilution and E-test strips for determination of the minimal inhibitory concentration (MIC) according to recommendations from the Brazilian Committee on Antimicrobial Susceptibility Testing (BrCAST) and European Committee on Antimicrobial Susceptibility Testing (EUCAST). Gene mecA characterization and Sccmec typing were performed by multiplex polymerase chain reaction (PCR) assay, and gene lukF detection by single PCR. Patients were prospectively followed up for two months, in order to determine their clinical characteristics and outcomes. Results. Overall, 48 Staphylococcus aureus isolates were obtained from 68 samples recovered from patients with Co-SMSI. Twenty two (42%) were phenotypically characterized as MRSA, although mecA gene was only identified in 20 of those samples. Sccmec was untypable in 12 isolates, Sccmec was type II in 4 isolates and 2 were classified as type IVa. LukF gene was identified in 5 isolates. Antimicrobial resistance profile showed that all isolates were susceptible to linezolid and vancomycin with MIC = 1 and MIC = 2 in 66,7% and 33.3%, respectively. Susceptibility to quinolones was worryingly low and none of the isolates were sensitive to usual doses of ciprofloxacin and levofloxacin, and showed increased rates of resistance to increased exposure to these drugs, as well. Isolates were both susceptible to gentamicin and tetracycline in 85% and resistance to also Sulfamethoxazole/Trimethoprim occurred in only 2 isolates. Mortality rate evaluated within 1 month of the initial evaluation was 10% among MRSA isolates. Conclusions. Our results showed that CA-MRSA isolates causing Co-SMSI demonstrated an alarming pattern of multidrug resistance, including to β-lactam and quinolones, which have been commonly prescribed as empirical therapy for patients with skin, soft tissue and musculoskeletal infections