Objectives. The purpose of this study was to examine the bactericidal efficacy of hydrogen peroxide (H. 2. O. 2. ) on Cutibacterium acnes (C. acnes). We hypothesize that H. 2. O. 2. reduces the bacterial burden of C. acnes. Methods. The effect of H. 2. O. 2. was assessed by testing bactericidal effect, time course analysis, growth inhibition, and minimum bactericidal concentration. To assess the bactericidal effect, bacteria were treated for 30 minutes with 0%, 1%, 3%, 4%, 6%, 8%, or 10% H. 2. O. 2. in saline or water and compared with 3% topical H. 2. O. 2. solution. For time course analysis, bacteria were treated with water or saline (controls), 3% H. 2. O. 2. in water, 3% H. 2. O. 2. in saline, or 3% topical solution for 5, 10, 15, 20, and 30 minutes. Results were analyzed with a two-way analysis of variance (ANOVA) (p < 0.05). Results. Minimum inhibitory concentration of H. 2. O. 2. after 30 minutes is 1% for H. 2. O. 2. prepared in saline and water. The 3% topical solution was as effective when compared with the 1% H. 2. O. 2. prepared in saline or water. The controls of both saline and water showed no reduction of bacteria. After five minutes of exposure, all mixtures of H. 2. O. 2. reduced the percentage of live bacteria, with the topical solution being most effective (p < 0.0001). Maximum growth inhibition was achieved with topical 3% H. 2. O. 2. . Conclusion. The inexpensive and commercially available topical solution of 3% H. 2. O. 2. demonstrated superior bactericidal effect as observed in the minimum bactericidal inhibitory concentration, time course, and colony-forming unit (CFU) inhibition assays. These results support the use of topical 3% H. 2. O. 2. for five minutes before surgical skin preparation prior to shoulder surgery to achieve eradication of C. acnes for the skin. Cite this article: P. Hernandez, B. Sager, A. Fa, T. Liang, C. Lozano, M. Khazzam. Bactericidal efficacy of hydrogen peroxide on Cutibacterium acnes. Bone Joint Res 2019;8:3–10. DOI: 10.1302/2046-3758.81.BJR-2018-0145.R1

Aims. Cutibacterium acnes (C. acnes; previously known as Propionibacterium acnes or P. acnes) periprosthetic hip and knee infections are under-reported. While culture contamination with C. acnes occurs, true infections are important to recognize and treat. We sought to describe the demographics and treatment outcomes of patients with C. acnes periprosthetic joint infections (PJIs) of the hip and knee. Methods. Patients with C. acnes PJI between January 2005 and December 2018 were retrospectively reviewed utilizing the institutional total joint registry. Patients with monomicrobial PJI and two or more positive cultures were considered to have true C. acnes PJI. Patients with polymicrobial infection or with only one positive culture were excluded. This resulted in 35 PJIs (21 hips and 14 knees); the patients’ mean age was 63 years (35 to 84) and 15 (43%) were female. Mean follow-up was five years (1 to 14). Results. The median time to positive culture was five days (IQR 5 to 6) and median synovial fluid cell count was 22,583 cells (IQR 15,200 to 53,231). The median ESR was 25 mm/hr (IQR 7 to 37), and CRP was 15 mg/l (IQR 3 to 29). Of the 35 PJIs, 18 (51%) were treated with chronic antibiotic suppression without surgical intervention, and the remainder were treated with two-stage exchange arthroplasty. The two-year survival free of any revision was 94%. Four patients failed treatment due to symptomatic infection, with three treated with two-stage exchange and one treated with irrigation and debridement with modular component exchange for a survival rate of 89% and 83% at two and five years, respectively. Conclusion. Laboratory evidence of C. acnes PJI in this cohort was typical compared to more conventional organisms. Cultures grew more quickly than previously thought in patients with C. acnes PJI. Treatment with two-stage exchange or chronic antibiotic suppression alone both had few treatment failures at mid-term follow-up. Cite this article: Bone Joint J 2024;106-B(12):1426–1430

Background. Data regarding the diagnostic value of ultrasound (US)-determined fluid film and joint aspiration prior to revision total hip arthroplasty (THA) for suspected periprosthetic joint infections (PJIs) is limited. This study aimed to analyse (1) the value of US-determined fluid film, (2) characterisation of the pre- and intraoperative microbiological spectrum and resistance patterns and (3) the concordance between preoperative synovial fluid and intraoperative culture results. Methods. We analysed 366 US-examinations from 340 patients prior to revision THA. Selected cases were categorized into clearly infected, non-infected and inconclusive, according to the International Consensus Meeting (ICM) 2018 Criteria. If US-determined fluid film was <1mm, no aspiration was performed based on our institutional standard protocol. Patients were grouped into no-aspiration (144/366;[39.3%]), dry-tap (21/366;[5.7%]) and a successful-tap (201/366;[54.9%]). The microbiological spectrum and antibiotic resistance patterns were determined and differences were compared between pre- and intraoperative results. Results. The absence of US-determined fluid film showed no correlation with the presence of hip PJI. Overall, 29.9% cases of the no-aspiration-group had a confirmed PJI. Discrepancies were found in 43.2% between successful taps and intraoperative cultures. The most prevalent microorganisms in preoperative synovial fluid were Staphylococcus epidermidis (20.9%), Staphylococcus aureus (20.9%) and Enterococcus faecalis (9.3%). The most prevalent microorganisms in intraoperative cultures were Staphylococcus epidermidis, Cutibacterium acnes and other coagulase-negative Staphylococci (14.2%). Additional microorganisms were identified in 43.8% intraoperatively. Staphylococcus aureus was more often detected preoperatively (20.9% vs. 5.8%;P=0.003), and Cutibacterium acnes intraoperatively (2.3% vs. 14.4%;P=0.01). There were no differences between the antibiotic resistance patterns of pre- and intraoperative concordant microorganisms. Conclusion. Absence of US-determined fluid film cannot rule out the presence of hip PJI. US-guided joint aspirations is a well-established technique. However, the preoperative analysis of synovial fluid shows high discrepancies especially in Cutibacterium acnes and other rare gram-positive microorganisms compared to intraoperative cultures

Aim. Low-grade infections are difficult to diagnose. As the presence of a chronic infection requires extensive surgical debridement and antibiotic treatment, it is important to diagnose a SII prior to surgery, especially when the hardware is revised. We investigated whether serum inflammatory markers or nuclear imaging can accurately diagnose a chronic spinal instrumentation infection (SII) prior to surgery. Method. All patients who underwent revision spinal surgery after a scoliosis correction between 2017 and 2019 were retrospectively evaluated. The diagnostic accuracy of serum C-reactive protein (CRP) and Erythrocyte Sedimentation Rate (ESR), . 18. F-fluorodeoxyglucose positron emission tomography combined with computed tomography (FDG-PET/CT) and Technetium-99m-methylene diphosphonate (99mTc-MDP) 3-phase bone scintigraphy (TPBS) to diagnose infection were studied. Patients with an acute infection or inadequate culture sampling were excluded. SII was diagnosed if ≥ 2 of the same microorganism(s) were isolated from intra-operative tissue cultures. Results. 31 patients were included. The indication for hardware extraction was pseudoarthrosis in the majority of patients (n = 15). 22 patients (71%) were diagnosed with SII. In all infected cases, Cutibacterium acnes was isolated, including 5 cases with a polymicrobial infection. Sensitivity, specificity, PPV and NPV was: 4.5%, 100%, 100% and 30.0% for CRP >10.0 mg/L, 5.5%, 100%, 100% and 29% for ESR > 30 mm/h; 56%, 80%, 83% and 50% for FDG-PET/CT and 50%, 100%, 100% and 20% for TPBS, respectively. Conclusions. The prevalence of SII in patients undergoing revision spinal surgery is high, with Cutibacterium acnes as the main pathogen. No diagnostic tests could be identified that could accurately diagnose or exclude SII prior to surgery. Future studies should aim to find more sensitive diagnostic modalities to detect low-grade inflammation

Aims. The outcomes of patients with unexpected positive cultures (UPCs) during revision total hip arthroplasty (THA) and total knee arthroplasty (TKA) remain unknown. The objectives of this study were to establish the prevalence and infection-free implant survival in UPCs during presumed aseptic single-stage revision THA and TKA at mid-term follow-up. Methods. This study included 297 patients undergoing presumed aseptic single-stage revision THA or TKA at a single treatment centre. All patients with at least three UPCs obtained during revision surgery were treated with minimum three months of oral antibiotics following revision surgery. The prevalence of UPCs and causative microorganisms, the recurrence of periprosthetic joint infections (PJIs), and the infection-free implant survival were established at minimum five years’ follow-up (5.1 to 12.3). Results. Of the 297 patients undergoing aseptic revisions, 37 (12.5%) had at least three UPCs obtained during surgery. The UPC cohort included 23 males (62.2%) and 14 females (37.8%), with a mean age of 71.2 years (47 to 82). Comorbidities included smoking (56.8%), hypertension (48.6%), diabetes mellitus (27.0%), and chronic renal impairment (13.5%). The causative microorganisms included Staphylococcus epidermidis (49.6%), Bacillus species (18.9%), Micrococcus species (16.2%), and Cutibacterium acnes (16.2%). None of the study patients with UPCs developed further PJIs or required further surgical intervention during follow-up. Conclusion. The prevalence of UPCs during presumed aseptic revision THA and TKA was 12.5%. The most common causative microorganisms were of low virulence, and included S. epidermidis, Bacillus species, Micrococcus species, and C. acnes. Microorganism-specific antibiotic treatment for minimum three months’ duration of UPCs in presumed aseptic revision arthroplasty was associated with excellent infection-free implant survival at mid-term follow-up. Cite this article: Bone Jt Open 2024;5(10):832–836

Aims. To evaluate the histopathological examination of peri-implant tissue samples as a technique in the diagnosis of postoperative spinal implant infection (PSII). Methods. This was a retrospective analysis. Patients who underwent revision spinal surgery at our institution were recruited for this study. PSII was diagnosed by clinical signs, histopathology, and microbiological examination of intraoperatively collected samples. Histopathology was defined as the gold standard. The sensitivity for histopathology was calculated. A total of 47 patients with PSII and at least one microbiological and histopathological sample were included in the study. Results. PSII occurred in approximately 28% of the study population. Histopathology showed a sensitivity of 51.1% in the diagnosis of PSII. The most commonly found pathogens were Cutibacterium acnes and gram-positive staphylococci. Conclusion. Histopathology has low sensitivity for detecting PSII. In particular, infections caused by low-virulence microorganisms are insufficiently detected by histopathology. Cite this article: Bone Joint J 2020;102-B(7):899–903

Aims. This study aimed to assess the performance of an automated multiplex polymerase chain reaction (mPCR) technique for rapid diagnosis of native joint septic arthritis. Patients and Methods. Consecutive patients with suspected septic arthritis undergoing aseptic diagnostic joint aspiration were included. The aspirate was used for analysis by mPCR and conventional microbiological analysis. A joint was classed as septic according to modified Newman criteria. Based on receiver operating characteristic (ROC) analysis, the area under the ROC curve (AUC) values of the mPCR and the synovial fluid culture were compared using the z-test. A total of 72 out of 76 consecutive patients (33 women, 39 men; mean age 64 years (22 to 92)) with suspected septic arthritis were included in this study. Results. Of 72 patients, 42 (58%) were deemed to have septic joints. The sensitivity of mPCR and synovial fluid culture was 38% and 29%, respectively. No significant differences were found between the AUCs of both techniques (p = 0.138). A strong concordance of 89% (Cohen’s kappa: 0.65) was shown. The mPCR failed to detect Staphylococcus aureus (n = 1) and Streptococcus pneumoniae (n = 1; no primer included in the mPCR), whereas the synovial fluid culture missed six microorganisms (positive mPCR: S. aureus (n = 2), Cutibacterium acnes (n = 3), coagulase-negative staphylococci (n = 2)). Conclusion. The automated mPCR showed at least a similar performance to the synovial fluid culture (the current benchmark) in diagnosing septic arthritis, having the great advantage of a shorter turnaround time (within five hours). Cite this article: Bone Joint J 2019;101-B:288–296

Aims. During revision procedures for aseptic reasons, there remains a suspicion that failure may have been the result of an undetected subclinical infection. However, there is little evidence available in the literature about unexpected positive results in presumed aseptic revision spine surgery. The aims of our study were to estimate the prevalence of unexpected positive culture using sonication and to evaluate clinical characteristics of these patients. Patients and Methods. All patients who underwent a revision surgery after instrumented spinal surgery at our institution between July 2014 and August 2016 with spinal implants submitted for sonication were retrospectively analyzed. Only revisions presumed as aseptic are included in the study. During the study period, 204 spinal revisions were performed for diagnoses other than infection. In 38 cases, sonication cultures were not obtained, leaving a study cohort of 166 cases. The mean age of the cohort was 61.5 years (. sd. 20.4) and there were 104 female patients. Results. Sonication cultures were positive in 75 cases (45.2%). Hardware failure was the most common indication for revision surgery and revealed a positive sonication culture in 26/75 cases (35%) followed by adjacent segment disease (ASD) in 23/75 cases (30%). Cutibacterium acnes and Staphylococcus epidermidis were the most commonly isolated microorganisms, observed in 45% and 31% of cases, respectively. C. acnes was isolated in 65.2% of cases when the indication for revision surgery was ASD. Conclusion. Infection must always be considered as a possibility in the setting of spinal revision surgery, especially in the case of hardware failure, regardless of the lack of clinical signs. Sonication should be routinely used to isolate microorganisms adherent to implants. Cite this article: Bone Joint J 2019;101-B:621–624

Aim. The origin of surgical site and biomaterial-associated infection is still elusive. Microorganisms contaminating the wound may come from the air, the surgical team, or from the skin of the patient. Prior to surgery the skin of patients is disinfected, but bacteria deeper in the skin (e.g. in sweat glands or sebaceous glands), may not be reached. This study aims to assess a potential role of this intracutaneous bacterial reservoir in biomaterial-associated infection. Method. To study if cutaneous microbiota colonize the wound when released from the skin upon cutting, we isolated, quantified and identified aerobic and anaerobic bacteria from the skin of 99 patients undergoing trauma surgery, before and after skin disinfection, from the knife blades and from the wound directly after the first cut. Results. Ninety-nine percent of the patients were culture-positive before disinfection with chlorhexidine. Of these, 40% were still culture-positive after disinfection. Of these, 54% had a positive culture of the wound after the skin cut. Twenty percent of the patients with a negative culture after disinfection, nevertheless had a positive wound culture after cutting the skin. Staphylococcus epidermidis and Cutibacterium acnes were the most often cultured bacterial species. In 9%, more than 100 bacterial colonies were cultured from the wound, a dose that may cause biomaterial-associated infections. Conclusions. Bacteria residing in the skin and not eradicated by disinfection may enter the surgical wound upon cutting, resulting in contamination which may cause a biomaterial-associated infection. Use of two knifes likely reduces the risk of wound contamination

Aim. Cutibacterium acnes is a major skin commensal that may also act as an opportunistic pathogen. Findings of C. acnes in tissue cultures obtained during arthroplasty revision surgery are difficult to interpret, since they may represent true infection or contamination. This study investigated whether C. acnes isolates obtained from prosthetic joint infections (PJIs) were related and shared common genomic traits that might correlate with clinical courses and patient outcomes. Method. C. acnes isolates from revision surgery of patients with PJIs of the hip, shoulder, and knee were characterized using molecular methods to determine sequence type (ST) and the presence of virulence determinants (CAMP factors, dermatan sulfate-binding adhesion 1, hyaluronidase lyase, and linear plasmid). A standardized review of the patients’ medical charts was performed. Results. The study included 37 patients with C. acnes culture-positive tissue samples where multiple isolates of C. acnes belonged to the same ST. Most of the isolates belonged to phylotype IA. 1. Phylogenetic analysis of virulence determinants revealed no shared pattern among PJI isolates. Seven patients had a polymicrobial infection. Exchange revision was performed in 70% of the patients, and >50% of all patients received antibiotic treatment for ≥3 months. Failure was noted in seven patients, all of whom had shoulder PJIs. Conclusions. No specific ST or any identifiable unique feature among virulence determinants were found among C. acnes isolated from PJIs of hips and shoulders. The majority of all included patients had low inflammatory markers and were treated successfully, even when the infection consisted of a polymicrobial infection

Aim. Accurate diagnosis is key in correctly managing prosthetic joint infection(PJI). Shoulder PJI definition and diagnosis is challenging. Current PJI definitions, based overwhelmingly in hip/knee research, may not accurately diagnose shoulder PJI. Our aim is to compare the preoperative performance of two PJI definitions comparing it to definitive postoperative classification. Method. This is a retrospective study of patients who have undergone total shoulder revision surgery for infection between 2005 and 2022. Cases were classified using two different PJI definitions: a)the European Bone and Joint Infection Society (EBJIS) and; 2)the 2018 International Consensus Meeting(ICM) PJI specific shoulder definition. Preoperative classification was based on clinical features, inflammatory markers and synovial fluid leukocyte count and definitive classification also considered microbiology and histology results. Results. Preoperative and definitive PJI classification status of the 21 patients included were evaluated and is summarized in table 1. The shoulder specific 2018 ICM definition showed the highest agreement between preoperative and definitive classification (76.2%, k=0.153, p=0.006) compared to EBJIS (52.4%, k=0.205, p=0.006). In all cases, the classification was changed because of positive intraoperative microbiology (at least two identical isolates). Microbiology findings showed coagulase negative staphylococci, Staphyloccocus aureus and Cutibacterium acnes to be the most frequent. Four patients had polymicrobial infections. Conclusions. Both the EBJIS 2021 and 2018 ICM definitions have low accuracy in predicting shoulder PJI preoperatively. Clearly further studies with larger cohorts are in dire need focusing specifically on shoulder revision arthroplasty to improve on existing definitions. Caution is advised while extrapolating of criteria/thresholds recommended for hip/knee joints. For any tables or figures, please contact the authors directly

Introduction. Multiple studies have identified Cutibacterium acnes (C.acnes) and other microbes in intervertebral disc tissue using 16S DNA Sequencing and microbial cultures. However, it remains unclear whether these bacteria are native to the discs or result from perioperative contamination. Our study aimed to detect Gram-positive bacteria in non-herniated human disc samples and explore correlations with Toll-like receptors (TLR) 2, TLR4, NLRP3, and Gasdermin D. Methods. Immunohistochemical staining was conducted on 75 human IVD samples for Gram-positive bacteria, S. aureus, C.acnes, TLR2, TLR4, NLRP3, and Gasdermin D. Cell detection and classification were performed using QuPath. NP cells were treated with Lipopolysaccharide (LPS) and Peptidoglycan (PGN) in monolayer and alginate beads for up to 72 hours, followed by secretome analysis using Luminex. Statistical analysis included Kruskal-Wallis, Dunn's multiple comparison test, and Pearson correlation. Results. Immunohistochemical staining revealed Gram-positive bacteria exclusively within cells, with C. acnes positivity ranging from 5–99% and correlating with patient age (r=0.41, p= 0.007). TLR2 positivity ranged from 5–99% and TLR4 from 3–72%, showing a strong correlation (r= 0.62, p= 1.5e-006). Females with mid-degenerative grades exhibited significantly decreased TLR2 expression compared to those without degeneration signs. Treatment with LPS and PGN increased catabolic cyto- and chemokines associated with IVD degeneration. Conclusion. In conclusion, this study confirms Gram-positive bacteria presence in non-herniated human disc samples and highlights their role in triggering a catabolic response in disc cells. No conflicts of interest.  . Sources of funding. This project is part of the Disc4All Training network to advance integrated computational simulations in translational medicine, applies to intervertebral disc degeneration and funded by Horizon 2020 (H2020-MSCA-ITN-ETN-2020 GA: 955735)

A key cause of low back pain is the degeneration of the intervertebral disc (IVD). Causality between infection of the IVD and its degenerative process gained great interest over the last decade. Granville Smith et al. (2021) identified 36 articles from 34 research studies investigating bacteria in human IVDs. Bacteria was identified in 27 studies, whereas 9 attributed bacterial presence to contamination. Cutibacterium acnes was the most abundant, followed by coagulase-negative staphylococcus. However, whether bacteria identified were present in vivo or represent perioperative contamination remains unclear. This study investigated whether bacteria are present in IVDs and what potential effects they may have on native disc cells. Immunohistochemical staining for Gram positive bacteria was performed on human IVD tissue to identify presence and characterise bacterial species. Nucleus pulposus (NP) cells in monolayer and 3D alginate were stimulated with LPS and Peptidoglycan (0.1-50 µg/ml) for 48hrs. Following stimulation qPCR for factors associated with disc degeneration including matrix genes, matrix degrading enzymes, cytokines, neurotrophic factors and angiogenic factors and conditioned media collected for ELISA and luminex analysis. Gram positive bacteria was detected within human IVD tissue. Internalisation of bacteria by NP cells influenced the cell and nuclei morphology. Preliminary results of exposure of NP cells to bacterial components indicate that LPS as well as Peptidoglycan increase IL-8 and ADAMTS-4 gene expression following 48 hours of stimulation with a dose response seen for IL-8 induction by peptidoglycan compared to the control group. Underlining these results, IL-8 protein release was increased for treated groups compared to non-treated control. Further analysis is underway investigating other output measures and additional biological repeats. This study has demonstrated bacteria are present within IVD cells within IVD tissue removed from degenerate IVD and is determining the potential influence of these on disc degeneration

Aims. Of growing concern in arthroplasty is the emergence of atypical infections, particularly Cutibacterium (formerly Propionibacterium) sp. infections. Currently, the dermal colonization rate of Cutibacterium about the hip is unknown. Therefore, the aim of this study was to investigate colonization rates of Cutibacterium sp. at locations approximating anterior and posterolateral approaches to the hip joint. Methods. For this non-randomized non-blinded study, 101 adult patients scheduled for hip or knee surgery were recruited. For each, four 3 mm dermal punch biopsies were collected after administration of anaesthesia, but prior to antibiotics. Prebiopsy skin preparation consisted of a standardized preoperative 2% chlorhexidine skin cleansing protocol and an additional 70% isopropyl alcohol mechanical skin scrub immediately prior to biopsy collection. Two skin samples 10 cm apart were collected from a location approximating a standard direct anterior skin incision, and two samples 10 cm apart were collected from a lateral skin incision (suitable for posterior, direct-lateral, or anterolateral approaches). Samples were cultured for two weeks using a protocol optimized for Cutibacterium. Results. A total of 23 out of 404 cultures (collected from 101 patients) were positive for a microorganism, with a total of 22 patients having a positive culture (22%). Overall, 15 of the cultures in 14 patients were positive for Cutibacterium sp. (65%), of which Cutibacterium acnes comprised the majority (n = 13; 87%). Other isolated microorganisms include coagulase-negative Staphylococcus (n = 6), Clostridium (n = 1), and Corynebacterium (n = 1). Of all positive cultures, 15 were obtained from the anterior location (65%), of which seven (60%) were from the most proximal biopsy location. However, these findings were not statistically significant (anterior vs lateral, p = 0.076; proximal vs distal, p =0.238). Conclusion. Approximately 14% (14/101) of the patients demonstrated a positive Cutibacterium colonization about the hip, the majority anteriorly. Given the high colonization rate of Cutibacterium, alternative skin preparations for total hip arthroplasty should be considered. Cite this article: Bone Joint J 2020;102-B(7 Supple B):52–56

Objectives. This study aims to investigate whether bacteria are present in intervertebral discs (IVDs) and their influence. Causality between chronic infection of the IVD and its degenerative process gained great interest recently. Granville Smith et al. (2021) identified 36 articles from 34 research studies investigating bacteria in IVDs, from these 27 studies found, Cutibacterium acnes being the most abundant. However, whether bacteria identified were present in vivo or if they represent contamination remains unclear. Methods. Human IVD tissue was fixed in paraffin and Immunohistochemical stained for Gram-positive bacteria. NP cells in monolayer have been stimulated with LPS (0.1–50 µg/ml) and Peptidoglycan (0.1–50 µg/ml) for 24, 48 and 72 hrs to investigate their influence. The concentration of proinflammatory and catabolic cytokines in the media is being measured using ELISA. RNA extracted and RT-qPCR utilised for factors associated with disc degeneration matrix genes, matrix degrading enzymes, cytokines, neurotrophic factors and angiogenic factors. Results. Bacteria were detected within IVD tissue. Bacteria was internalized by the NP cells and influenced the nuclei morphology. Preliminary results of the exposure of NP cells to bacterial components demonstrate that ADAMTS4 as well as IL-8 were showed an increase in gene expression after LPS and peptidoglycan treatment compared to the non-treated control. Underlining these results, IL-8 protein was increased in treated groups, whereas peptidoglycan treated groups showed a dose dependence. Conclusion. This study demonstrates that Gram positive bacteria are present within the IVD. The exposure of NP cells to peptidoglycans indicates that bacterial components trigger a stress response. Conflicts of Interest: No conflict of interest. Sources of Funding: This project is part of the Disc4All Training network to advance integrated computational simulations in translational medicine, applies to intervertebral disc degeneration and funded by Horizon 2020 (H2020-MSCA-ITN-ETN-2020 GA: 955735)

Aim. In 10% of the presumed aseptic hip or knee revisions, a low-grade infection is unexpectedly diagnosed based on the tissue samples taken during revision. Extended antimicrobial prophylaxis can possibly reduce the failure rate in cases of unexpected PJI, because the prophylaxis can be considered as early empiric treatment. In this randomized controlled study we analysed whether extended antimicrobial prophylaxis compared to a single dose is beneficial to improve the outcome of treatment in unexpected PJI in revision arthroplasty. Method. This study was nested in a randomized clinical trial comparing single-dose cefazolin with prolonged prophylaxis (15 doses of cefazolin over 5 days) for revision arthroplasty of the hip or knee. For this analysis, patients were included if an unsuspected PJI (defined as ≥2 positive intraoperative tissue samples with the same microorganism) was diagnosed. PJI treatment consisted of 12 weeks of a rifampicin-based regimen in Staphylococcal PJI, without removal of the prosthesis. We examined Infection characteristics and success of treatment after one year, defined as the absence of signs or treatment for PJI during follow-up. Results. After randomization of 662 patients, 68 unexpected PJI were diagnosed. In 5 cases no antimicrobial treatment was started. The success rate after one year follow-up for those who received PJI treatment was 96% (28/29) in the single dose group and 91% (31/34) in the extended prophylaxis group (p=1.00). The most frequently identified pathogens in unexpected PJI were Cutibacterium acnes (n=50) and Staphylococcus epidermidis (n=14). The causatives were susceptible for the cefazolin prophylaxis in 61 of the 63 cases. The interval between the stopped prophylaxis and the re-start of antimicrobial treatment was on average 10 days (SD 4) for the single dose and 5 days (SD 4) for the extended group. The mean duration of antimicrobial treatment was 83 days (SD 12) and did not differ between both groups (p=0.16). Conclusions. This is the first randomized controlled trial in which extended prophylaxis showed no benefit on the prosthesis survival for patients with an unexpected PJI after assumed aseptic revision of the hip or knee prosthesis. The results imply that extended prophylaxis should not be given as part of early empiric therapy

The diagnosis of infection following shoulder arthroplasty is notoriously difficult. The prevalence of prosthetic shoulder infection after arthroplasty ranges from 3.9 – 15.4% and the most common infective organism is Cutibacterium acnes. Current preoperative diagnostic tests fail to provide a reliable means of diagnosis including WBC, ESR, CRP and joint aspiration. Fluoroscopic-guided percutaneous synovial biopsy (PSB) has previously been reported in the context of a pilot study and demonstrated promising results. The purpose of this study was to determine the diagnostic accuracy of percutaneous synovial biopsy compared with open culture results (gold standard). This was a multicenter prospective cohort study involving four sites and 98 patients who underwent revision shoulder arthroplasty. The cohort was 60% female with a mean age was 65 years (range 36-83 years). Enrollment occurred between June 2014 and November 2021. Pre-operative fluoroscopy-guided synovial biopsies were carried out by musculoskeletal radiologists prior to revision surgery. A minimum of five synovial capsular tissue biopsies were obtained from five separate regions in the shoulder. Revision shoulder arthroplasty was performed by fellowship-trained shoulder surgeons. Intraoperative tissue samples were taken from five regions of the joint capsule during revision surgery. Of 98 patients who underwent revision surgery, 71 patients underwent both the synovial biopsy and open biopsy at time of revision surgery. Nineteen percent had positive infection based on PSB, and 22% had confirmed culture positive infections based on intra-operative tissue sampling. The diagnostic accuracy of PSB compared with open biopsy results were as follows: sensitivity 0.37 (95%CI 0.13-0.61), specificity 0.81 (95%CI 0.7-0.91), positive predictive value 0.37 (95%CI 0.13 – 0.61), negative predictive value 0.81 (95%CI 0.70-0.91), positive likelihood ratio 1.98 and negative likelihood ratio 0.77. A patient with a positive pre-operative PSB undergoing revision surgery had an 37% probability of having true positive infection. A patient with a negative pre-operative PSB has an 81% chance of being infection-free. PSB appears to be of value mainly in ruling out the presence of peri-prosthetic infection. However, poor likelihood ratios suggest that other ancillary tests are required in the pre-operative workup of the potentially infected patient

Aim. There is growing evidence that bacteria encountered in periprosthetic joint infections (PJI) form surface-attached biofilms on prostheses, as well as biofilm aggregates embedded in synovial fluid and tissues. However, models allowing the investigation of these biofilms and the assessment of their antimicrobial susceptibility in physiologically relevant conditions are currently lacking. To address this, we developed a synthetic synovial fluid (SSF) model and we validated this model in terms of growth, aggregate formation and antimicrobial susceptibility testing, using multiple PJI isolates. Methods. 17 PJI isolates were included, belonging to Staphylococcus aureus, coagulase negative staphylococci, Cutibacterium acnes, Pseudomonas aeruginosa, enterococci, streptococci, Candida species and Enterobacterales. Growth and aggregate formation in SSF, under microaerophilic or anaerobic conditions, were evaluated using light microscopy. The biofilm preventing concentration (BPC) and minimum biofilm inhibitory concentration (MBIC) of relevant antibiotics (doxycyclin, rifampicin and oxacillin) were determined for the staphylococcal strains (n=8). To this end, a high throughput approach was developed, using a fluorescent viability resazurin staining. BPC and MBIC values were compared to the minimum inhibitory concentration (MIC) obtained with conventional methods. Results. The SSF model allowed all isolates to grow well under microaerophilic or anaerobic conditions. When cultured in SSF, all isolates formed biofilm aggregates, varying in size and shape along different species. A susceptibility testing method based on measuring resazurin-derived fluorescence was successfully developed, allowing high throughput determination of the BPC and the MBIC in SSF. For all staphylococci cultured in SSF a reduced susceptibility to the tested antibiotics was observed when compared to susceptibility data obtained in general medium. For rifampicin and doxycyclin the BPC was consistently higher than the MIC (two- to fourfold dilution difference for rifampicin and four- to sixfold dilution difference for doxycyclin). For oxacillin the MIC equaled the BPC for two isolates, while for the other isolates the BPC was higher than the MIC (two- to fourfold dilution difference). Expectedly, the MBIC was higher than the BPC and differences with the MIC were even more pronounced for all antibiotics tested (differences of six- to fourteenfold dilutions were observed). Conclusion. Our data indicate that the in vitro SSF model could provide more insight in how PJI-related pathogens form biofilms in physiologically relevant conditions. The BPC and MBIC were consistently and substantially higher than MIC. This model could be a valuable addition to evaluate the antimicrobial susceptibility in biofilms in a PJI context. Sources of funding: FWO-Vlaanderen (grant G066523N)

Aim. Treatment algorithms for fracture-related nonunion depend on the presence or absence of bacterial infection. However, the manifestation of septic nonunion varies. Low-grade infections, unlike manifest infections, lack clinical signs of infection and present similarly to aseptic nonunion. The clinical importance of low-grade infection in nonunion is not entirely clear. Therefore, the aim of this study was to evaluate the clinical relevance of low-grade infection in the development and management of femoral or tibial nonunion. Method. A prospective, multicenter clinical study enrolled patients with nonunion and regular healed fractures. Preoperatively, complete blood count without differential, C-reactive protein (CRP), and procalcitonin were obtained, clinical signs of infection were recorded, and a suspected septic or aseptic diagnosis was made based on history and clinical examination. During surgical nonunion revision or routine implant removal, tissue samples were collected for microbiology and histopathology, and osteosynthesis material for sonication. Nonunion patients were followed for 12 months. Definitive diagnosis of “septic” or “aseptic” nonunion was made according to diagnostic criteria for fracture-related infection, considering the results of any further revision surgery during follow-up. Results. 34 patients with regular healed fractures were included. 62 nonunion patients were diagnosed as aseptic, 22 with manifest, and 23 with low-grade infection. The positive predictive value was 88% and the negative predictive value 72% for the suspected diagnosis. The nonunion groups had significantly higher CRP levels than the regular healer group. Differentiation between septic and aseptic nonunion based on blood values was not possible. Low-grade infection demonstrated less frequently histopathologic signs of infection than manifest infection (22% vs. 50%, p=0.048), with 15% of regular healers having histopathologic signs of infection. Cutibacterium acnes was less present in manifest compared to low-grade infection (p=0.042). Healing rates for septic nonunion involving C. acnes were significantly lower for manifest infection (20%) than for low-grade infection (100%, p=0.002). Patients with low-grade infection were treated with systemic antibiotics less frequently than patients with manifest infection (p=0.026), with no significant difference in healing rate (83% vs. 64%), which was slightly lower for low-grade infection than for aseptic nonunion (90%). Conclusions. Low-grade infections play a significant role in nonunion development and are difficult to diagnose preoperatively due to the lack of clinical signs of infection and unremarkable blood counts. However, our results imply that for low-grade infections, antibiotic therapy may not always be mandatory to heal the nonunion. This study was supported by the German Social Accident Insurance (FF-FR0276)