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

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

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

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

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

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.