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

Ruling out an infection in one-stage knee and hip revisions for presumed aseptic failure by conventional tissue cultures takes up to 14 days. Multiplex polymerase chain reaction (PCR) is a quick test (4–5 hours) for detecting infections. The purpose of this study was to evaluate the negative predictive value of an automated multiplex PCR for the detection of microorganisms in synovial fluid obtained intraoperatively in unsuspected knee and hip revisions. The NPV of the multiplex PCR U-ITI system of synovial fluid compared to tissue cultures of knee and hip revisions was 95.7% and 92.5%, respectively. Cultures required several days for growth whereas the automated mPCR U-ITI system provided results within five hours. The multiplex PCR U-ITI system is a quick and reliable test in ruling out infection in presumed aseptic knee and hip revisions. With this test the number of unsuspected infected revisions can be lowered and antibiotic overtreatment as well as undertreatment after one-stage revision arthroplasty can be avoided. This directly results in a reduction in length of hospital stay, hospital costs and possible antibiotic resistance development

Differentiating cases of aseptic loosening of total hip arthroplasty (THA) from loosening due to low-grade infection can often be difficult. It is possible that some cases of ‘aseptic’ loosening may be related to unidentified bacterial infection. Using Polymerase Chain Reaction (PCR), this study attempted to identify the frequency with which bacterial DNA could be observed at revision arthroplasty for what was considered ‘aseptic’ loosening. All revision cases had to fulfil strict criteria to be considered aseptically loose In all cases operative specimens from the synovial fluid, synovium, femoral and acetabular membranes where possible were sent for analysis by histology, bacteriology and by PCR to identify the presence of the 16S bacterial ribosomal fraction, an indicator of bacterial DNA. Ten bacteria per millilitre of tissue/fluid were the threshold for detection. As a control for environmental contamination, specimens from primary THA were also sent for analysis in the same manner as revisions. The identification of bacterial DNA in at least one sample from a patient was considered a positive case result. 45 revision THA were identified over a 3-year period (1998–2001). From those 45 revision cases, 163 specimens were sent for analysis by PCR. These specimens were compared to the control group of 34 primary THA from which 91 specimens were sent for analysis by PCR. When analysed by specimens positive by PCR, bacterial DNA was identified in 55 of 163 specimens sent from the 45 revision THA. This compared with 21 of 91 specimens positive by PCR sent from the 34 primary THA (p=0. 07). When analysed by cases positive by PCR, bacterial DNA was identified in 29 of 45 revision THA and in 8 of 34 primary THA (p< 0. 001). PCR is a sensitive test for detecting infection in revision THA. Results from the primary THA cases would suggest there is at least a 23% false positive rate even with negative bacterial culture. The increased frequency with which bacterial DNA has been identified in ‘aseptically’ loose revision THAs, however, is unlikely to be due solely to environmental contamination. These results may have relevance for our interpretation and understanding of aseptic loosening as well for the diagnosis of prosthetic infection

Pre-revision detection of infection in failed total joint replacements (TJR) is essential to allow appropriate management planning. Unfortunately, low-grade infection is often difficult to detect. The use of molecular biology may offer increased sensitivity in this setting. We have analysed the use of the Polymerase Chain Reaction (PCR) to diagnose infection in pre-operative aspirates in a group of patients undergoing revision arthroplasty. We prospectively tested 50 aspirates in 50 patients with failed TJR (34 hips and 16 knees). Antibiotics were omitted for 2 weeks prior to aspiration. The aspirate was sent for microbiological culture in aerobic and anaerobic conditions. An aliquot was retained for PCR analysis which involved DNA extraction then amplification of an 882 base pair segment of the Universal 16S RNA gene. In 33 patients who subsequently underwent revision arthroplasty multiple specimens were taken from around the joint for microbiological and histological examination and the presence or absence of pus was noted. The patient was deemed to be infected if one of these criteria was found: 2 or more intra-operative cultures positive for the same organism; an acute inflammatory response on histology; pus in the joint at revision . 1. . PCR was positive in 29 cases. Aspiration microbiology was positive in 13 cases. Of the 33 cases revised, 15 patients were deemed to be infected using the previously established criteria, described above. Compared to preoperative aspiration microbiology PCR had a sensitivity of 92% and specificity of 54%. Compared to the published criteria for infection, PCR was 93% sensitive and 61% specific. If rheumatoid cases are excluded the specificity improves to 71%. It was concluded that PCR has the ability to amplify very small amounts of target DNA. The apparently high false positive rate compared to aspiration microbiology may indicate that PCR is picking up DNA from contaminating or non-viable organisms (treated or phagocytosed), giving poor specificity. However, microbiology is known to have poor sensitivity on pre-operative aspiration samples, and some of the microbiology results may be false negative. Compared to the criteria for infection after revision our results for PCR are more encouraging, especially for non-rheumatoid patients. These patients are part of an ongoing study to identify the most reliable criteria for pre-operative diagnosis of infection in total joint replacement

Low virulent chronic prosthetic infection might be indistinguishable to aseptic loosening. Polymerase chain reaction (PCR) has been introduced to improve bacterial detection of implant infection. However, there is great risk of false positive results when using broad range/universal PCR primers. The source of DNA contaminants may be of environmental origin as well as the reagents employed. To study the presence of bacterial DNA in culture negative biopsy specimens by using defined criteria for PCR positivity. We included six specimens from each of 21 patients preoperatively considered having aseptic loosening of a hip prosthesis. These 126 specimens were culture negative after seven days of incubation. Prior to incubation, the specimens were divided, and half of each specimen was processed for PCR. Three sets of primer pairs targeting the 16S rRNA gene were developed. Nine specimens from culture proven prosthetic infections and nine specimens from primary prosthetic surgery served as positive and negative controls, respectively. A specimen was considered PCR positive if:. bacterial DNA ≥ 2 times than the reagent control, and. a positive PCR signal for ≥ 2 primer pairs. All the 126 patient specimens were PCR negative and the nine positive controls were positive. One of nine negative specimen controls was PCR positive, DNA sequencing demonstrated a non-pathogen. Five single PCR reactions (1.3 %) were positive. This study underlines the importance of establishing stringent criteria for interpretation of PCR positivity to apply to clinical specimens. By the criteria used, five single positive PCR reactions were identified as false positives. Positive PCR reactions in all of the specimen positive controls prove the detection ability of the method. One PCR positive result in a specimen negative control demonstrates the contamination risk in collection and handling of biopsies. In conclusion, by using a semiquantitative PCR and stringent criteria for PCR positivity we were unable to detect any infections missed by culture

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

Introduction: Previous in vivo kinematic studies have assessed total knee arthroplasty (TKA) motion under weight-bearing conditions. This in vivo study analyzed and compared posterior cruciate retaining (PCR) and posterior stabilized (PS) kinematics under passive and weight-bearing conditions in subjects implanted with both a PCR and PS TKA. Methods: Eighteen subjects were implanted with a PCR and a PS TKA, by a single surgeon using a similar surgical technique. Both implant designs had similar condylar geometry. Femorotibial contact positions for all 18 subjects (PCR and PS), implanted by a single surgeon, were analyzed using video fluoroscopy. Each subject,while under fluoroscopic surveillance, performed a weight-bearing deep knee bend and a passive, nonweight-bearing flexion. Video images were downloaded to a workstation computer and analyzed at varying degrees of knee flexion. Femorotibial contact paths for the medial and lateral condyles, axial rotation and femoral condylar lift-off were then determined using a computer automated model-fitting technique. Femorotibial contact anterior to the tibial midline in the sagittal plane was denoted as positive and contact posterior was denoted as negative. Results: Under passive and weight-bearing conditions, the PCR TKA experienced more paradoxical anterior translation than the PS TKA. Under passive, non weight-bearing conditions, the PS TKA, on average, experienced 3.5 mm of posterior femoral rollback, compared to only 0.6 mm for the PCR TKA. Under weight-bearing conditions, the PS TKA experienced only 0.6 mm of posterior femoral rollback, compared to 0.9 mm for the PCR TKA. The maximum anterior slide was 10.0 mm for the PCR TKA and only 2.7 mm for the PS TKA. There was greater variability in both the PCR and PS anteroposterior data. Subjects having a PCR TKA experienced more normal axial rotation patterns. Sixteen of 18 PCR TKA experienced a normal axial rotation pattern under weight-bearing conditions, while only 9/18 PS TKA experienced a normal pattern. Nonweight-bearing, passive axial rotation patterns were more abnormal for both groups than the weight-bearing patterns. The greatest difference between passive and weight-bearing conditions occurred in the condylar lift-off data. Under passive conditions, both TKA groups experienced significantly greater magnitude and incidence of condylar lift-off. The maximum amount of condylar lift-off under passive conditions was 5.0 mm for the PCR TKA and 6.4 mm for the PS TKA. Discussion: This is the first in vivo kinematic study to assess a comparison between PCR and PS TKA implanted by the same surgeon in the same patient. Subjects in this study experienced more abnormal kinematic patterns, especially condylar lift-off, when tested under passive, nonweight-bearing conditions. Subjects having a PS TKA experienced less variability in their kinematic data, but PCR TKA, on average, experienced more normal axial rotation and less condylar lift-off

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

Introduction: Infection is a serious complication of joint arthroplasty. Detection of low-grade prosthetic infection can be difficult, with major implications on the subsequent treatment, cost and patient morbidity. We evaluated the effectiveness of Polymerase Chain Reaction (PCR) in detecting infection in patients undergoing arthroplasty revision surgery. Methods: Ninety-one consecutive patients (92 joints) undergoing revision THA or TKA were assessed prospectively. Preoperative assessment included clinical examination, blood tests and plain radiographs. At revision, tissue samples were sent for microbiology and histology. Cultures, using blood culture bottles, and PCR were performed on the synovial fluid. Diagnosis of infection relied on the surgeon’s opinion encompassing the clinical presentation, the results of various investigations and the intraoperative findings. Infected arthroplasties underwent a 2-stage revision. Post-operatively patients were followed up at regular intervals for a minimum of 2 years. Results: Twelve (13%) joints were infected. Histology was positive for infection in 11 cases, tissue cultures were positive in 12 and PCR was positive in 32 cases. Intraoperative tissue cultures had sensitivity 0.75, specificity 0.96, positive predictive value 0.75 and negative predictive value 0.96; histology had sensitivity 0.92, specificity 1, positive predictive value 1 and negative predictive value 0.99 and PCR had a sensitivity 0.92, specificity 0.74, positive predictive value 0.34 and negative predictive value 0.98. At 2 years no patient showed evidence of infection. Discussion: PCR is a sensitive method of diagnosing prosthetic infection but has poor specificity. False positive results may be due to contamination in theatre or in the laboratory. Positive results in apparently non-infected cases could be due to the detection of low virulence organisms, a small number of bacteria or a strong host immune response. Bacterial fragments and non-culturable forms of bacteria may also be responsible. Conclusion: PCR was not helpful as a screening test for prosthetic infection. Cultures and histology combined with the surgeon’s clinical judgment remain the gold standard

Introduction. The MITCH PCR is an anatomic, flexible, horse-shoe shaped acetabular component, with 2 polar fins. The rationale of the PCR cup design is to reproduce a near-physiological stress distribution in the bone adjacent to the prosthesis. The thin composite cup is designed to fuse and flex in harmony with the surrounding bony structure. Only the pathological acetabular cartilage and underlying subchondral bone of the horseshoe-shaped, load-bearing portion of the acetabular socket is replaced, thus preserving viable bone stock. The PCR is manufactured from injection moulded carbon fibre reinforced polyetheretherketone (PEEK), with a two layer outer surface comprising hydroxyapatite and plasma sprayed commercially pure titanium. It is implanted in conjunction with a large diameter low wear femoral head, producing a bearing that will generate minimal wear debris with relatively inert particles. Pre-clinical mechanical testing, finite element analysis and biocompatibility studies have been undertaken. FEA evaluation predicts preservation of host bone density in the load bearing segments. A pilot clinical study was completed on a proto-type version of the PCR cup (the “Cambridge” cup), achieving excellent 5 and 10 year results. Subjects and Methods. We report the three-year results from a two-centre, prospective clinical evaluation study of the MITCH PCR cup. Patient outcome has been assessed using standardised clinical and radiological examinations and validated questionnaires. The change in physical level of activity and quality of life has been assessed using the Oxford Hip Score, Harris Hip score and the EuroQol-5D score, at scheduled time-points. Serial radiographs have been analysed to monitor the fixation and stability of the components. Results and Conclusions. In total 25 PCR cups were implanted by 3 surgeons. There were 12 men and 13 women. The mean patient age at time of surgery was 67 years (range 57–74). An Accolade TMZF stem was used as the femoral component in 19 patients and an Exeter stem in 6. The mean Oxford Hip score improved from 19.8 pre-operatively to 45 at the latest follow-up. The mean Euroqol-5D score improved from 62.6 to 83.6 and the Harris Hip score improved from 49.9 to 90.6. Three adverse events were noted in 2 patients (2 chest infections and 1 deep vein thrombosis). One revision of the acetabular component was performed at 21 months for squeaking. This has been investigated and modification of the articular geometry has resolved the problem on in-vitro testing

Introduction: Aseptic loosening of THR has a multifactorial aetiology. Differentiating such cases from loosening due to low-grade infection can often be difficult. It is possible that at least some cases of ‘aseptic’ loosening may be related to unidentified bacterial infection. This study attempted to identify the frequency with which bacterial DNA could be observed in the periprosthetic membrane and synovial fluid of patients undergoing revision surgery for what was considered ‘aseptic’ loosening. Methods: Specimens from 39 revision and 31 primary hip replacements were obtained. The latter were used as a control for environmental contamination. All revision THR cases were investigated pre-operatively for infection by CRP, ESR, WCC, Gallium Scan. Operative specimens were analysed by bacteriological culture as well as by PCR to identify the presence of the 16S bacterial ribosomal fraction. Results were analysed by Chi square test. Results: By PCR, bacterial DNA was identified in 22 of 39 revision hip surgery specimens and 6 of 31 primary hip replacement specimens (p=0.002). By culture none of these specimens had any bacterial growth. Conclusions: The increased frequency with which bacterial DNA has been identified in ‘aseptically’ loose revision THR is unlikely to be due solely to environmental contamination although this remains a concern. These results may have relevance for our interpretation and understanding of aseptic loosening as well for the diagnosis of prosthetic infection

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

Introduction: Our group has previously reported on microarray gene expression profiling of failed aseptic and septic THRs. The data obtained from the Affymetrix DNA chips suggested a range of 21 differentially expressed genes between the tissue samples obtained from the control and study patients with failed aseptic THRs. The variation in expression that was demonstrated did not suggest that the basis of the local tissue reaction that occurs in aseptic loosening of THR is primarily inflammatory in nature. In order to validate these results we have performed quantitative real-time polymerase chain reaction (RT-PCR) to analyse the transcriptional levels of genes expression in the samples used in our original study and to formulate a hypothesis of how these candidate genes can be related to aseptic join loosening. Methods: 3 control and 6 aseptic samples of peri-prosthetic membrane were subjected to RNA extraction. RNA quality analysis and quantification were performed. SYBRâ Green I real time quantitative PCR (RT qPCR) assays were designed using Primer Express [Applied Biosystems] and BLAST searching the resulting sequences. The comparative method for quantitation of gene expression levels, which utilizes arithmetic formulas to give the similar results to those achieved with standard curves, was utilised to validate the cDNA microarray data. Results: We were able to devise successful quantitative real-time PCR for 15 of the 21 candidate genes plus the reference gene GAPDH. The genes coding for complement component C4B, Osteonectin , ATP2A2 (an ATPase linked to the regulation of adhesion, differentiation and proliferation in tissue that expresses this gene such as bone) and Phospholipase2A, were all found to be under-expressed whereas SLC2A5 (a solute carrier that can facilitate glucose/fructose transport)and NPC1 (intimately involved in cholesterol and glycolipid trafficking and inversely related to PLA2-mediated release of eicosanoids such as PGE2) were found to be over-expressed. Conclusions: The data from our gene expression and RT-PCR studies have suggested novel pathways that may be intimately involved in the development of peri-prosthetic osteolysis and aseptic loosening that are distinctly different from the currently accepted theory of a proinflammatory cytokine cascade initiated by tissue reaction to particulate wear debris. These include possible alteration in both extra- and intracellular Ca2+ metabolism together with a possible effect upon extra-cellular matrix function. Altered lipid metabolism may also be evident and in particular decreased eicosanoid production. Intriguingly, the pattern of gene expression that is seen our studies would appear to be quite different than that seen in synovial inflammatory arthritidies such as rheumatoid and osteo-arthritis and suggests that previous studies that has used these pathological mechanisms as comparisons or controls may be flawed

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.

Background. Both from experimental studies and the large arthroplasty registries there is evidence that bacteria are more often involved in implant loosening then is currently reported. To further elucidate this potential problem, the current study investigated the hypothesis that many total hip arthroplasty revisions, classified as aseptic, are in fact low-grade infections missed with routine diagnostics. Methods. In 7 Dutch hospitals, 176 patients with the preoperative diagnosis of aseptic loosening of their total hip arthroplasty were enrolled. From each patient, the preoperative history was obtained. During surgery, between 14 and 20 tissue samples were obtained for routine culture, pathology analysis and broad range 16S rRNA PCR with reverse line blot hybridization (PCR-RLB). Samples were taken from the (neo-) capsule and acetabular and femoral interface tissue. Cultures were performed locally according to similar protocols. One specialized pathologist, blinded for all other results, analyzed all pathology samples. The PCR-RLB analysis was performed centrally, using a technique previously validated for orthopedic use. Patients were classified as not infected, suspect for infection or infected, according to strict, predefined criteria. Each patient had a follow-up visit after 1 year. Results. Seven patients were classified as infected, of whom 4 were not identified by routine culture. In these patients, positive PCR-RLB results were supported by pathology analyses suspect for infection as well. An additional 15 patients were suspect for infection as well. The microorganisms identified were low virulent bacteria, like coagulase negative staphylococci and Proprionibacterium acnes, in most cases. Twenty of these 22 patients received a cemented prosthesis, fixated with antibiotic-loaded bone cement. All patients received prophylactic systemic antibiotics, after obtaining the tissue samples. Seven of the 22 patients reported complaints one year post-surgery, only one showing signs of early loosening. However, in none of the patients additional surgery was performed. Discussion. Although percentages were not as high as previously reported in literature, between 4 and 13 percent of patients with the preoperative diagnosis of aseptic loosening were infected. However, as thorough debridement was performed during surgery and prophylactic antibiotics were used, it did not have many clinical consequences, as most patients performed well at the 1-year follow-up. Whether it has implications for long-term implant survival remains to be seen

The lack of an accurate, rapid diagnostic test for mycobacterium tuberculosis (TB) is a major handicap in the management of spinal TB. GeneXpert, a new, rapid molecular diagnostic test is recommended as the first line investigation for suspected pulmonary TB in areas with a high prevalence of HIV or drug resistance, yet it has not been validated for the diagnosis of musculoskeletal TB.

The aim of this study was to assess the accuracy of GeneXpert in diagnosing spinal TB.

A prospective clinical study of 69 consecutive adults with suspected spinal TB was conducted at a tertiary hospital in an area with the highest incidence and prevalence of TB in the world. GeneXpert was used on tissue samples of the enrolled patients and its diagnostic accuracy compared with a reference standard of tissue in liquid culture. A total of 71 spine samples from 69 patients (two re-biopsies) were included in the study.

The GeneXpert test showed a sensitivity of 95.6% and specificity of 96.2% for spinal TB. The results of the GeneXpert test were available within 48 hours compared with a median of 35 days (IQR 15 to 43) for cultures. All cases of multi-drug resistant TB (MDR TB) were diagnosed accurately with the GeneXpert test. The MDR TB rate was 5.8%.

Cite this article: Bone Joint J 2014;96-B:1366–9.

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.