Aims

The diagnosis of periprosthetic joint infection can be difficult due to the high rate of culture-negative infections. The aim of this study was to assess the use of next-generation sequencing for detecting organisms in synovial fluid.

Aim. Diagnosing Orthopaedic infection is limited by the sensitivity of culture methods. Next generation sequencing (NGS) offers an alternative approach for detection of microorganisms from clinical specimens. However, the low ratio of pathogen DNA to human DNA often inhibits detection of microorganisms from specimens. Depletion of human DNA may enhance the detection of microbial DNA. 1. Our aim was to compare four DNA extraction methods for the recovery of microbial DNA from orthopaedic samples for NGS. Method. Simulated samples; pooled culture negative sample matrix was spiked with known concentrations of microorganisms, each panel consisting of 7 samples. Broth culture was performed on simulated samples for comparison with NGS. *. . DNA Extraction; total nucleic acid extraction was performed on an automated extraction platform. **. using the viral NA assay. Modifications included: (1) mechanical lysis (glass beads), (2) lysis of human cells (saponin 0.025%), turbo DNase treatment and (3) mechanical lysis and addition of MspJI enzyme post-extraction for methylated DNA digestion. Detection of human and microbial DNA; human endogenous (HE) gene rtPCR. ***. was utilised following manufacturer's recommendations. Microbial DNA was detected using SYBR green 16s ribosomal RNA rtPCR with high resolution melt-curve analysis. ****. . Results. Broth culture recovered 64% (9/14) of the microorganisms from simulated samples. A significant increase (p<0.01) in the cycle threshold (C. T. ) (median C. T. 25.9 IQR 25.5, 26.1) of the HE gene rtPCR was observed using extraction method b, indicating a significant reduction in human DNA. No significant change (p=0.38) in the C. T. of the HE gene rtPCR was observed between the baseline method (median C. T. 19.2 IQR 18.5, 19.7) and modifications a (median C. T. 18.4 IQR 18.2, 19.4) and c (median C. T. 19.3 IQR 18.6, 19.4). Detection of microbial DNA was successful using the base line extraction method and modification a. Microbial DNA was not detected using the 16s ribosomal RNA rtPCR for modifications b and c. Conclusions. This study has demonstrated that modification of DNA extraction methods using selective enzymatic digestion of human DNA negatively impacts on the recovery of microbial DNA from simulated specimens. Total DNA extraction allows the successful recovery of microbial DNA alongside a significant amount of human DNA. The effect of the presence of human DNA will be subsequently assessed through NGS CosmosID analysis to establish if NGS is more sensitive than broth based culture

Introduction

Surgical management of PJI remains challenging with patients failing treatment despite the best efforts. An important question is whether these later failures reflect reinfection or the persistence of infection. Proponents of reinfection believe hosts are vulnerable to developing infection and new organisms emerge. The alternative hypothesis is that later failure is a result of an organism that was present in the joint but was not picked up by initial culture or was not a pathogen initially but became so under antibiotic pressure. This multicenter study explores the above dilemma. Utilizing next-generation sequencing (NGS), we hypothesize that failures after two stage exchange arthroplasty can be caused by an organism that was present at the time of initial surgery but not isolated by culture.

Identification of modalities and procedures to improve the differential diagnosis of septic and aseptic cases in patients with joint-related pain after total hip or knee alloplasty (THA/TKA). A prospective cohort of 147 patients presenting with problems related to previous THA or TKA was included and subjected to a comprehensive diagnostic algorithm. The standard diagnostics were supplemented with novel or improved methods for sampling of clinical specimens, sonication of retrieved implant parts, prolonged and effective culture of microorganisms, and dedicated clinical samples for molecular biological detection and identification of microorganisms. Furthermore, comprehensive pain investigations and nuclear imaging were employed. For each case the clinical management was decided upon in a clinical conference with participation of clinical microbiologist, orthopedics and experts in nuclear imaging. The clinical management of patients was blinded against the molecular biological detection of microorganisms. Patients grouped as follows: 69 aseptic, 19 acute septic, 19 chronic septic, 40 pain/unresolved. Sonication of retrieved implant parts resulted in detection of biofilm not detected by standard specimens, i.e. joint fluid and periprosthetic tissue biopsies. Next generation sequencing detected and identified few infections not detected by culture. Molecular analyses showed more polymicrobial infections than culture. Nuclear imaging was inconclusive with respect to recommendation of changed setup. Analysis of blood based biomarkers is ongoing. Patients with chronic pain are undergoing follow-up. The special emphasis put on detection of infections resulted in detection of infections in joints that otherwise would have been categorized as aseptic loosening. Clinical management for these cases was changed accordingly. The cross-disciplinary clinical conference is considered valuable for clinical management. The clinical relevance of the polymicrobial nature of infections as diagnosed employing next generation sequencing is yet to be established. Long-term follow-up is planned

Aims. Current diagnostic tools are not always able to effectively identify periprosthetic joint infections (PJIs). Recent studies suggest that circulating microRNAs (miRNAs) undergo changes under pathological conditions such as infection. The aim of this study was to analyze miRNA expression in hip arthroplasty PJI patients. Methods. This was a prospective pilot study, including 24 patients divided into three groups, with eight patients each undergoing revision of their hip arthroplasty due to aseptic reasons, and low- and high-grade PJI, respectively. The number of intraoperative samples and the incidence of positive cultures were recorded for each patient. Additionally, venous blood samples and periarticular tissue samples were collected from each patient to determine miRNA expressions between the groups. MiRNA screening was performed by small RNA-sequencing using the miRNA next generation sequencing (NGS) discovery (miND) pipeline. Results. Overall, several miRNAs in plasma and tissue were identified to be progressively deregulated according to ongoing PJI. When comparing the plasma samples, patients with a high-grade infection showed significantly higher expression levels for hsa-miR-21-3p, hsa-miR-1290, and hsa-miR-4488, and lower expression levels for hsa-miR-130a-3p and hsa-miR-451a compared to the aseptic group. Furthermore, the high-grade group showed a significantly higher regulated expression level of hsa-miR-1260a and lower expression levels for hsa-miR-26a-5p, hsa-miR-26b-5p, hsa-miR-148b-5p, hsa-miR-301a-3p, hsa-miR-451a, and hsa-miR-454-3p compared to the low-grade group. No significant differences were found between the low-grade and aseptic groups. When comparing the tissue samples, the high-grade group showed significantly higher expression levels for 23 different miRNAs and lower expression levels for hsa-miR-2110 and hsa-miR-3200-3p compared to the aseptic group. No significant differences were found in miRNA expression between the high- and low-grade groups, as well as between the low-grade and aseptic groups. Conclusion. With this prospective pilot study, we were able to identify a circulating miRNA signature correlating with high-grade PJI compared to aseptic patients undergoing hip arthroplasty revision. Our data contribute to establishing miRNA signatures as potential novel diagnostic and prognostic biomarkers for PJI. Cite this article: Bone Jt Open 2024;5(6):479–488

Hip resurfacing may be a useful surgical procedure when patient selection is correct and only implants with superior performance are used. In order to establish a body of evidence in relation to hip resurfacing, pseudotumour formation and its genetic predisposition, we performed a case-control study investigating the role of HLA genotype in the development of pseudotumour around MoM hip resurfacings. All metal-on-metal (MoM) hip resurfacings performed in the history of the institution were assessed. A total of 392 hip resurfacings were performed by 12 surgeons between February 1st 2005 and October 31st 2007. In all cases, pseudotumour was confirmed in the preoperative setting on Metal Artefact Reduction Sequencing (MARS) MRI. Controls were matched by implant (ASR or BHR) and absence of pseudotumour was confirmed on MRI. Blood samples from all cases and controls underwent genetic analysis using Next Generation Sequencing (NGS) assessing for the following alleles of 11 HLA loci (A, B, C, DRB1, DRB3/4/5, DQA1, DQB1, DPB1, DPA1). Statistical significance was determined using a Fisher's exact test or Chi-Squared test given the small sample size to quantify the clinical association between HLA genotype and the need for revision surgery due to pseudotumour. Both groups were matched for implant type (55% ASR, 45% BHR in both the case and control groups). According to the ALVAL histological classification described by Kurmis et al., the majority of cases (63%, n=10) were found to have group 2 histological findings. Four cases (25%) had group 3 histological findings and 2 (12%) patients had group 4 findings. Of the 11 HLA loci analysed, 2 were significantly associated with a higher risk of pseudotumour formation (DQB1*05:03:01 and DRB1*14:54:01) and 4 were noted to be protective against pseudotumour formation (DQA1*03:01:01, DRB1*04:04:01, C*01:02:01, B*27:05:02). These findings further develop the knowledge base around specific HLA genotypes and their role in the development of pseudotumour formation in MoM hip resurfacing. Specifically, the two alleles at higher risk of pseudotumour formation (DQB1*05:03:01 and DRB1*14:54:01) in MoM hip resurfacing should be noted, particularly as patient-specific genotype-dependent surgical treatments continue to develop in the future

Aim. Periprosthetic joint infection is an increasing reason for revision surgery. Tissue cultures are a standard (std.) diagnostic procedure but may be hindered by bacteria that are difficult to cultivate. The use of dithiothreitol (DTT) to detach the formed biofilm has been proposed to improve the diagnostic security. The aim was to compare the diagnosis results using the microDTTect device with the routine PJI diagnostics and next generation sequencing (NGS) from DTT treated explants. Method. 66 patients with revision surgeries were included in this study (38 aseptic; 28 septic). We compared std. microbiology tissue cultures with the microDTTect cultures of the DTT treated explants and NGS of bacterial DNA isolated from DTT solution. Results. In 75% of the septic cases, the std. microbiology was in line with the microDTTect cultures. In 8% of the aseptic cases, the microDTTect culture indicated a present pathogen. In 71% of the septic cases, NGS was compared to the std. microbiology and NGS. The concordance in the aseptic cohort between NGS and std. microbiology was 79%. Staphylococcus were most frequently detected by all three techniques Polymicrobial infections, were detected less frequently by culturing techniques, but with a high sensitivity using NGS. Conclusion. Our data indicate that tissue cultures show a similar reliability compared to the other techniques. The DTT culture method had a sensitivity of 75% while the specificity was 92%. NGS had a sensitivity of 71% and a specificity of 79%. These results may improve the treatment decision in clinical practice

Summary Statement. Using the latest Next Generation Sequencing technologies, we have investigated miRNA expression profiles in human trabecular bone from total hip replacement (THR) revision surgery where wear particle associated osteolysis was evident. Introduction. A major problem in orthopaedic surgery is aseptic loosening of prosthetic implants caused by wear particle associated osteolysis. Wear debris is known to impact on a variety of cellular responses and genes in multiple pathways associated with the development of the periprosthetic osteolysis. MicroRNAs (miRNAs) act as negative regulators of gene expression and the importance of miRNAs in joint pathologies has only recently been addressed. However, miRNA profiles in osteolytic bone are largely unknown. Using the latest Next Generation Sequencing technologies, we have investigated miRNA expression profiles in human trabecular bone sourced from bone discarded during total hip replacement (THR) revision surgery where wear particle associated osteolysis was evident. Patients and Methods. Three groups of gender and age-matched patients (n=9 per group) were recruited for this study including patients undergoing revision surgery, primary THR patients and healthy subjects. Total RNAs were prepared from trabecular bone specimens. The cDNA libraries were constructed using a TruSeq Small RNA Sample Preparation kit, and then sequenced on an Illumina HiSeq2000 sequencer. All good quality tags were aligned against the reference sequences containing human chromosomal sequences and 18s and 28s rRNA sequences were analysed using Bowtie software. We used miRBase v19 to identify the start positions of all mature miRNA and the edgeR package to analyse differential expression. Osteogenesis pathway-related gene expression was also investigated using RT-qPCR Array assay. Results. We observed a significant difference in expressed miRNAs between revision and primary THR groups, including upexpressed miR127, miR-409, miR-211 and miR-146a. Importantly, the miR-127 (3.1 fold, p=0.005) and miR-146a (3.5 fold, p=0.001) were not only upexpressed in the revision group vs primary group, but also upexpressed in the revision group vs the healthy group. Thus, miR-127 and miR-146a may have potential as both biomarkers to predict osteolysis and as therapeutic targets. The miR-127 and miR-146a are critical in bone diseases because some of their target genes play an important role in osteogenesis. We have thus studied osteogenic genes and confirmed that SMAD4, RUNX2, FGFR1, TGFβ1, COL1A1 and WNT4 were downregulated. Our data also revealed that miR-93 and miR-204a were downexpressed (−3.7 fold, p=0.023; −2.5, p=0.003 respectively) and t IL-6 and IL-6R, which had been reported as miR-204 target genes, were upexpressed. Discussion and Conclusion. Our results showed that upexpressed miR-127, miR-146a, miR-204a and miR-93 in trabecular bone from revision THR may be the key negative regulators in either osteogenic genes involved in osteogenic differentiation of bone formation or inflammatory genes involved in osteoclastogenesis. Aberrant miRNA expressions identified in the revision THR group may also suggest the existence of genetic risk factors favouring the development of osteolysis in certain specific subgroups of patients. An in-depth understanding of the roles of these regulatory miRNAs in the skeleton warrants further investigation

Introduction. Recent focus has shifted towards the utilization of deoxyribonucleic acid (DNA) sequencing modalities in periprosthetic joint infection (PJI) diagnosis and organism identification. The purpose of this study was to compare the diagnostic accuracy of next generation sequencing (NGS) to polymerase chain reaction (PCR) multiplex, culture, the Musculoskeletal Infection Society (MSIS) criteria, and the recently proposed criteria by Parvizi et al. [1] in the diagnosis of periprosthetic hip infections. Methods. In this retrospective study, aspirate or tissue samples were collected in 23 revision and 19 primary hip arthroplasties for routine diagnostic workup for PJI and sent to the laboratory for NGS and PCR multiplex. Concordance along with statistical differences between diagnostic studies were calculated using Chi-squared test for categorical data. Results. When comparing to the MSIS criteria, concordance was 71.4% for NGS, 64.3% for PCR, and 92.9% for culture (p<0.001). There was no significant difference based on prior infection (p=0.695), or sample collection method (tissue swab or synovial fluid) (p=0.327). Seven samples were culture positive and NGS negative all of which met both criteria for PJI. Four patients were culture negative but NGS positive, of which 1 (25.0%) met both criteria. Concordance was 100% between the MSIS criteria and criteria proposed by Parvizi et al. [1]. Conclusion. In this initial cohort NGS was more accurate than 16s subunit PCR techniques, but less accurate than culture in the diagnosis of PJI determining the presence or absence of PJI. What is not clear is how NGS will perform against culture in terms of identifying the specific bacterial strain. Currently, laboratory tests used for either criteria for PJI diagnosis should be obtained regardless of NGS along with the overall clinical picture to help guide decision making for PJI treatment. For any tables or figures, please contact the authors directly

Introduction. Recent focus has queried whether of deoxyribonucleic acid (DNA) sequencing modalities of bacterial DNA found in periarticular fluid and tissues will improve in periprosthetic joint infection (PJI) diagnosis and organism identification diagnostic accuracy for periprosthetic joint infection The purpose of this study was to compare the diagnostic accuracy of next generation sequencing (NGS) to polymerase chain reaction (PCR) multiplex, and culture, the Musculoskeletal Infection Society (MSIS) criteria, and the recently proposed criteria by Parvizi et al. [1] in the diagnosis of periprosthetic knee infections. Methods. In this retrospective study, aspirate or tissue samples were collected in 70 revision and 58 primary knee arthroplasties for routine diagnostic workup for PJI and sent to the laboratory for NGS and PCR multiplex. Concordance along with statistical differences between diagnostic studies were calculated using Chi-squared test for categorical data. Results. When comparing to the MSIS criteria, concordance was 78.1% for NGS, 66.4% for PCR, and 85.9% for culture (p<0.001). There was no significant difference based on prior infection (p=0.825), or sample collection method (tissue swab or synovial fluid) (p=0.986). Fifteen samples were culture positive and NGS negative, of which 10 (66.7%) met both criteria for PJI. Thirteen patients were culture negative but NGS positive, of which 2 (15.4%) met both criteria. Concordance was 100% between the MSIS criteria and criteria proposed by Parvizi et al. [1]. Conclusion. In this initial cohort NGS was more accurate than 16s subunit PCR techniques, but less accurate than culture in the diagnosis of PJI determining the presence or absence of PJI. What is not clear is how NGS will perform against culture in terms of identifying the specific bacterial strain. Currently, laboratory tests used for either criteria for PJI diagnosis should be obtained regardless of NGS along with the overall clinical picture to help guide decision making for PJI treatment. For figures, tables, or references, please contact authors directly

Introduction. We aimed to identify genes associated with the development of ALVAL at relatively low levels of wear. Methods. At our unit all patients undergoing revision of a MoM hip prosthesis have periprosthetic tissue samples graded for ALVAL. Explants undergo volumetric wear testing of the bearing and taper surfaces. We identified patients with moderate/severe ALVAL who had been exposed to lower than the median wear rate of all recorded patients who had developed ALVAL (<3mm. 3. /year). This was termed the “ALVAL” group. We then identified all patients whose tissues had shown no signs of ALVAL. The patients in the two groups were sent buccal DNA collection kits. DNA was examined using next generation sequencing. Alleleic frequencies in the two groups were compared using Fisher's test and compared to a background UK population group (n=8514). We then conducted binary logistic regression with patient age, sex, primary source of debris (taper/bearing) and HLA genotype as the predictors. With the hypothesis that a cobalt/albumin metalloprotein acts as the epitope, we used validated binding prediction software to determine the relative affinities of the binding grooves created by different DQA1/DQB1 genetic combinations for albumin derived peptides. Given the protection that male sex and younger age appears to confer against ALVAL, we hypothesized that testosterone peptides may compete for these binding sites. Results. 28 ALVAL and 37 non ALVAL patients returned their samples for testing. The frequencies of DQA1∗05:05 and DQB1∗03:01 were significantly greater in the ALVAL groups(p=0.018). The variables positively associated with ALVAL were female sex(0.021), increasing age(0.003) and DQA1/DQB1 combinations with greater binding affinity for albumin fragments(0.03). Greater binding affinities for testosterone peptides were inversely related to ALVAL(0.05). Discussion. Common immune genotypes are associated with a greater risk of ALVAL. Conclusion. The evidence base on which MoM follow up protocols are based should be re-evaluated in light of these findings and future studies designed accordingly

Background. Recent reports demonstrate that Next Generation Sequencing (NGS) facilitates pathogen identification in the context of culture-negative PJI; however the clinical relevance of the polymicrobial genomic signal often generated remains unknown. This study was conceived to explore: (1) the ability of NGS to identify pathogens in culture-negative PJI; and (2) determine whether organisms detected by NGS, as part of a prospective observational study, had any role in later failure of patients undergoing surgical treatment for PJI. Methods. In this prospective study samples were collected in 238 consecutive patients undergoing revision total hip and knee arthroplasties. Of these 83 patients (34.9%) had PJI, as determined using the Musculoskeletal Infection Society (MSIS) criteria, and of these 20 were culture-negative (CN-PJI). Synovial fluid, deep tissue and swabs were obtained at the time of surgery and sent for NGS and culture/MALDI-TOF. Patients undergoing reimplantation were excluded. Treatment failure was assessed using the previously described Delphi criteria. In cases of re-operation, organisms present were confirmed by culture and MALDI-TOF. Concordance of the infecting pathogen(s) at failure with the NGS analysis at the initial stage CN- PJI procedure was determined. Results. Twenty cases of culture-negative PJI were identified (Figure 1). CNPJI rate in our samples was 24%. NGS was positive in 18 cases. Two cases were both culture and NGS negative. Eight CN-PJIs (8/20; 40%) failed by re-operation with infection recurrence confirmed on culture. In 7 of these 8 cases (88%), the organism at failure was present on NGS at the time of the initial CN-PJI procedure. The remaining case failed with a new organism, via likely hematogenous seeding from an inter-current infection (Figure 2). NGS detected several organisms in CN-PJI cases (Figure 3). Discussion. CN-PJI is often associated with polymicrobial genomic organism profile. Furthermore, most of the failures by infection recurrence were due to an organism previously detected by NGS. Our findings suggest some cases of PJI may be polymicrobial and escape detection using conventional culture. Further multi-institutional work with larger numbers and longer clinical follow-up is required for validation

Introduction. The prevalence of symptomatic osteoarthritis (OA) in the knee is 11–11% compared to 3.4–4.4% in the ankle. In addition to this, 70% of ankle arthritis is post-traumatic while the vast majority of knee arthritis is primary OA. Several reports have previously implicated biochemical differences in extracellular matrix composition between these joint cartilages; however, it is unknown whether there is an inherent difference in their transcriptome and how this might affect their respective functionality under load, inflammatory environment etc. Therefore, we have analysed the transcriptome of ankle and knee cartilage chondrocytes to determine whether this could account for the lower prevalence and altered aetiology of ankle OA. Methods. Human full-depth articular cartilage was taken from the talar domes (n=5) and the femoral condyles (n=5) following surgical amputation. RNA was extracted and next generation sequencing (NGS) performed using the NextSeq®500 system. Statistical analysis was performed to identify differentially regulated genes (p adj < 0.05). Data was analysed using Integrated Pathway Analysis software and genes of interest validated by quantitative PCR. Results. 809 genes were differentially expressed in this NGS study: 781 genes were significantly up-regulated and 27 significantly down-regulated in ankle cartilage with respect to knee. Preliminary analysis has identified several pathways which are differentially regulated including ‘inflammation mediated by cytokines’, ‘glutamate receptor pathway, ‘heterotrimeric-G-protein signalling pathways’, ‘WNT signalling’ and ‘integrin signalling’. Discussion. This is the first report identifying genes that are differentially expressed in ankle cartilage compared to the knee. Validation is currently being performed to ascertain the importance of these gene changes and correlation with their protein expression in the different joints. An understanding of the inherent biological differences in the cartilage between these two joints will provide invaluable insight into why the ankle is relatively spared from primary OA and the majority of ankle arthritis occurs following trauma

Introduction. Next generation sequencing (NGS) has been shown to facilitate detection of microbes in a clinical sample, particularly in the setting of culture-negative periprosthetic joint infection (PJI). However, it is unknown whether every microbial DNA signal detected by NGS is clinically relevant. This multi-institutional study was conceived to 1) identify species detected by NGS that may predict PJI, then 2) build a predictive model for PJI in a developmental cohort; and 3) validate the predictive utility of the model in a separate multi-institutional cohort. Methods. This multicenter investigation involving 15 academic institutions prospectively collected samples from 194 revision total knee arthroplasties (TKA) and 184 revision hip arthroplasties (THA) between 2017–2019. Patients undergoing reimplantation or spacer exchange procedures were excluded. Synovial fluid, deep tissue and swabs were obtained at the time of surgery and shipped to MicrogenDx (Lubbock, TX) for NGS analysis. Deep tissue specimens were also sent to the institutional labs for culture. All patients were classified per the 2018 Consensus definition of PJI. Microbial DNA analysis of community similarities (ANCOM) was used to identify 17 candidate bacterial species out of 294 (W-value >50) for differentiating infected vs. noninfected cases. Logistic Regression with LASSO model selection and random forest algorithms were then used to build a model for predicting PJI. For this analysis, ICM classification was the response variable (gold standard) and the species identified through ANCOM were the predictor variables. Recruited cases were randomly split in half, with one half designated as the training set, and the other half as the validation set. Using the training set, a model for PJI diagnosis was generated. The optimal resulting model was then tested for prediction ability with the validation set. The entire model-building procedure and validation was iterated 1000 times. From the model set, distributions of overall assignment rate, specificity, sensitivity, positive predictive value (PPV) and negative predicative value (NPV) were assessed. Results. The overall predictive accuracy achieved in the model was 75.9% (Figure 1). There was a high accuracy in true-negative and false-negative classification of patients using this predictive model (Figure 2), which has previously been a criticism of NGS interpretation and reporting. Specificity was 97.1%, PPV was 75.0%, and NPV was 76.2%. On comparison of the distribution of abundances between ICM-positive and ICM-negative patients, Staphylococcus aureus was the strongest contributor (F=0.99) to the predictive power of the model (Figure 3). In contrast, Cutibacterium acnes was less predictive (F=0.309) and noted to be abundant across both infected and noninfected revision TJA samples. Discussion. This study is the first to utilize predictive modeling algorithms on a large prospective multicenter database in order to transform analytic NGS data into a clinically relevant diagnostic signal. Our collaborative findings suggest the microbial DNA signal identified on NGS may be an independent useful adjunct for the diagnosis of PJI, as well as help identify causative organisms. Further work applying artificial intelligence tools will improve accuracy, predictive power and clinical utility of high-throughput sequencing technology. For figures, tables, or references, please contact authors directly

Recent evidence suggests that the microbial community, its spatial distribution and activity play an important role in the prolongation of treatment and healing of chronic infections. Standard bacterial cultures often underestimate the microbial diversity present in chronic infections. This lack of growth is often due to a combination of inadequate growth conditions, prior usage of antibiotics and presence of slow-growing, fastidious, anaerobic or unculturable bacteria living in biofilms. Thus, diagnosis of chronic infections is challenged by lack of appropriate sampling strategies and by limitations in microbiological testing methods. The purpose of this study was to improve sampling and diagnosis of prosthetic joint infections (PJI) and chronic wounds, especially considering the biofilm issue. Systematic sampling, sonication of prosthesis and extended culture were applied on patients with chronic wounds and patients with suspected PJIs. Optimized DNA extraction, quantitative PCR, cloning, next generation sequencing and PNA FISH were applied on the different types of specimens for optimized diagnosis. For further investigation of the microbial pathogenesis, in situ transcriptomics and metabolomics were applied. In both chronic wounds and PJIs, molecular techniques detected a larger diversity of microorganisms than culture methods in several patients. Especially in wounds, molecular methods identified more anaerobic pathogens than culture methods. A heterogeneous distribution of bacteria in various specimens from the same patient was evident for both patient groups. In chronic wounds, multiple biopsies from the same ulcer showed large differences in the abundance of S. aureus and P. aeruginosa at different locations. Transcriptomic and metabolomic analyses indicated the important virulence genes and nutrient acquisition mechanisms of Staphylococcus aureus in situ. As an example, diagnosis and treatment of a patient with a chronic biofilm prosthesis infection persisting for 7 years will be presented. Our studies show that diagnosis of chronic biofilm related infections required multiple specimen types, standardized sampling, extended culture and molecular analysis. Our results are useful for improvement of sampling, analysis and treatment in the clinic. It is our ambition to translate studies on bacterial activity into clinical practice in the future

Aims

Hip resurfacing remains a potentially valuable surgical procedure for appropriately-selected patients with optimised implant choices. However, concern regarding high early failure rates continues to undermine confidence in use. A large contributor to failure is adverse local tissue reactions around metal-on-metal (MoM) bearing surfaces. Such phenomena have been well-explored around MoM total hip arthroplasties, but comparable data in equivalent hip resurfacing procedures is lacking. In order to define genetic predisposition, we performed a case-control study investigating the role of human leucocyte antigen (HLA) genotype in the development of pseudotumours around MoM hip resurfacings.

Aims

The aim of this study was to analyze the prevalence of culture-negative periprosthetic joint infections (PJIs) when adequate methods of culture are used, and to evaluate the outcome in patients who were treated with antibiotics for a culture-negative PJI compared with those in whom antibiotics were withheld.

Aims

We aimed to report the mid- to long-term rates of septic and aseptic failure after two-stage revision surgery for periprosthetic joint infection (PJI) following total hip arthroplasty (THA).