Aims. Bacterial infection activates neutrophils to release neutrophil extracellular traps (NETs) in bacterial biofilms of periprosthetic joint infections (PJIs). The aim of this study was to evaluate the increase in NET activation and release (NETosis) and haemostasis markers in the plasma of patients with PJI, to evaluate whether such plasma induces the activation of neutrophils, to ascertain whether increased NETosis is also mediated by reduced DNaseI activity, to explore novel therapeutic interventions for NETosis in PJI in vitro, and to evaluate the potential diagnostic use of these markers. Methods. We prospectively recruited 107 patients in the preoperative period of prosthetic surgery, 71 with a suspicion of PJI and 36 who underwent arthroplasty for non-septic indications as controls, and obtained citrated plasma. PJI was confirmed in 50 patients. We measured NET markers, inflammation markers, DNaseI activity, haemostatic markers, and the thrombin generation test (TGT). We analyzed the ability of plasma from confirmed PJI and controls to induce NETosis and to degrade in vitro-generated NETs, and explored the therapeutic restoration of the impairment to degrade NETs of PJI plasma with recombinant human DNaseI. Finally, we assessed the contribution of these markers to the diagnosis of PJI. Results. Patients with confirmed PJI had significantly increased levels of NET markers (cfDNA (p < 0.001), calprotectin (p < 0.001), and neutrophil elastase (p = 0.022)) and inflammation markers (IL-6; p < 0.001) in plasma. Moreover, the plasma of patients with PJI induced significantly more neutrophil activation than the plasma of the controls (p < 0.001) independently of tumour necrosis factor alpha. Patients with PJI also had a reduced DNaseI activity in plasma (p < 0.001), leading to a significantly impaired degradation of NETs (p < 0.001). This could be therapeutically restored with recombinant human DNaseI to the level in the controls. We developed a model to improve the diagnosis of PJI with cfDNA, calprotectin, and the start tail of TGT as predictors, though cfDNA alone achieved a good prediction and is simpler to measure. Conclusion. We confirmed that patients with PJI have an increased level of NETosis in plasma. Their plasma both induced NET release and had an impaired ability to degrade NETs mediated by a reduced DNaseI activity. This can be therapeutically restored in vitro with the approved Dornase alfa, Pulmozyme, which may allow novel methods of treatment. A combination of NETs and haemostatic biomarkers could improve the diagnosis of PJI, especially those patients in whom this diagnosis is uncertain. Cite this article: Bone Joint J 2024;106-B(9):1021–1030

We examined the usefulness of neutrophil CD64 expression in detecting local musculoskeletal infection and the impact of antibiotics on its expression. Of 141 patients suspected of musculoskeletal infection, 46 were confirmed by microbiological culture to be infected and 95 had infection excluded. The median CD64 count of patients with localised infection was 2230 molecules per cell (interquartile range (IQR) 918 to 4592) and that of the patients without infection was 937 molecules per cell (IQR 648 to 1309) (p < 0.001). The level of CD64 correlated with the CRP level in patients with infection, but not in those without infection (r = 0.59, p < 0.01). Receiver operator characteristic curve analysis revealed that CD64 was a good predictor of local infection. When the patients were subdivided into two groups based on the administration of antibiotics at the time of CD64 sampling, the sensitivity for detecting infection was better in those who had not received antibiotics. These results suggest that measurement of CD64 expression is a useful marker for local musculoskeletal infection

Aim. Identifying the optimal agent for irrigation for periprosthetic joint infection remains challenging as there is limited data. The ideal solution should have minimal cytotoxicity while maintaining bactericidal activity. We developed a novel activated-zinc solution containing zinc-chloride (ZnCl. 2. ) and sodium-chlorite (NaClO. 2. ). The purpose of this study was 1.) to investigate the antimicrobial efficacy of 2 concentrations (“CZ1”, “CZ2”) against Staphylococcus aureus and Pseudomonas aeruginosa and 2.) to evaluate untoward effects of the solution on local wound tissue 24 hours after solution exposure in pig wound models. Method. The study was conducted and reported in accordance to ARRIVE guidelines. We created twenty-four 1.5cm wounds on the back of a Yorkshire-cross pig. Wounds were inoculated with standardized Pseudomonas and S. aureus. 8 wounds were designated as controls (inoculum without treatment), 8 treated with CZ1, and 8 with CZ2. Punch biopsies were taken 1 hour after treatment and bacteria quantified. Wound necrosis/neutrophil infiltrate was measured 24-hours post-exposure. Results. After 1-hour, the control, CZ1 and CZ2 wounds had total bacteria of 5.7, 2.8 and 3.5 logCFU/g, respectively (p=0.017). The control, CZ1 and CZ2 wounds had S. aureus of 5.3, 2.3 and 1.6 logCFU/g, respectively (p=0.009). The control, CZ1 and CZ2 wounds had Pseudomonas of 5.5, 0.3 and 0.0 logCFU/g, respectively (p=0.000). After 24 hours of exposure to CZ1 and CZ2, there was no statistically significant increased necrosis (p=0.12, p=0.31, respectively). CZ1 had increased, moderate neutrophil infiltrate (p=0.04) when compared to controls, however CZ2 was not significant (p=0.12). Conclusions. Our novel solution demonstrated 99.5–99.9% reduction in total bacteria, 99.9–99.98 % reduction in S. aureus, and 100% eradication of Pseudomonas 1-hour after exposure, without significantly increased necrosis and no-to-minimally-increased neutrophil infiltrate. This novel solution may provide another significant tool in the arsenal to treat and/or prevent PJI and other wound infections

Aim. To describe the histopathology of the first and last debrided bone tissue in chronic osteomyelitis and answer the following research question; is the last debrided bone tissue viable and without signs of inflammation?. Method. In total, 15 patients with chronic osteomyelitis were allocated to surgical treatment using a one stage protocol including extensive debridement. Suspected infected bone tissue eradicated early in the debridement procedure was collected as a clearly infected sample (S1). Likewise, the last eradicated bone tissue was collected as a suspected non-infected sample (S2), representing the status of the bone void. In all cases, the surgeon debrided the bone until visual confirmation of healthy bleeding bone. The samples were processed for histology, i.e. decalcification and paraffin embedding, followed by cutting and staining with Haematoxylin and Eosin. Immunohistochemistry with MAC-387 antibodies towards the calprotectin of neutrophil granulocytes (NGs) was also performed and used for estimation of a neutrophil granulocyte (NG) score (0, 1, 2 or 3), by the method described for fracture related infections (1). Results. For the S1 samples the median NG score was 3 which is considered confirmatory for infection. However, following debridement the median NG score was significantly (p = 0.032) reduced to 2. Often NGs were seen as single cells, but in seven S1 samples and in one S2 sample massive NG accumulations were observed. The S1 samples showed a mix of granulation tissue, fibrosis, viable bone, and bone necrosis. The S2 samples contained viable bone tissue and occasionally (10/15) small fragments of necrotic bone or bone debris were seen. Furthermore, a large number of erythrocytes were observed in most S2 samples. Conclusions. The present study shows that the inflammatory response still existents after debridement, although the response fades from the center of infection. Therefore, sampling of debrided bone tissue for histology must be performed initially during surgery, to avoid underestimation of the inflammatory response, i.e. the NG score. The last debrided bone tissue cannot by definition be considered completely viable and caution should be made to remove blood (rinse) before intraoperative evaluation of the viability of debrided cancellous bone. Remnant necrotic bone fragments or debris could represent low-vascular hiding places for leftover bacteria. Application of local antibiotics might have a central role in clearing of these small non-viable bone pieces at the bone void interface

Aim. Although established serum inflammatory biomarkers, such as serum C-reactive protein (CRP) and serum white blood cell count (WBC), showed low accuracies in the literature, they are still commonly used in diagnosing periprosthetic joint infections (PJI). For a sufficient preoperative diagnosis novel more accurate serum parameters are needed. The aim of our study was to evaluate the performances of the established and novel routinely available serum parameters in diagnosing periprosthetic joint infections when using the proposed European Bone and Joint Infection Society (pEBJIS) criteria. Method. In this retrospective study, 177 patients with an indicated revision surgery after a total joint replacement were included from 2015 to 2019. The easily accessible and routinely available serum parameters CRP, WBC, the percentage of neutrophils (%N), the neutrophils to lymphocytes ratio (NLR), fibrinogen and the platelet count to mean platelet volume ratio (PC/mPV) were evaluated preoperatively. The performances were examined via receiver operating characteristic (ROC) curve analysis (AUC). The curves were compared using the z-test. Seventy-five cases (42%) showed a PJI based on the pEBJIS-criteria. Results. The sensitivities of serum CRP (cut-off: ≥10mg/L), WBC (≥10×10^9 cells/L), %N (≥69.3%), NLR(≥ 3.82), fibrinogen (≥ 457 mg/dL), and PC/mPV (≥ 29.4) were calculated with 68% (95% CI: 57–78), 36% (26 – 47), 66% (54 – 76), 63% (51 – 73), 69% (57 – 78), and 43% (32 – 54), respectively. Specificities were 87% (79 – 93), 89% (81 – 94), 67% (57 76), 73% (63 – 81), 89% (80 – 93), and 81% (72 – 88), respectively. Serum CRP and fibrinogen showed better performances than the other evaluated serum parameters (p<0.0001). The median serum CRP (17.6 mg/L) in patients with PJI caused by a low virulence microorganism was lower compared with infections caused by high virulence organisms (49.2 mg/L; p=0.044). Synovial fluid leucocyte count and histology showed better accuracies than serum CRP, serum WBC, %N, NLR, serum fibrinogen, and PC/mPV (p<0.0001). Conclusions. Although serum CRP and fibrinogen showed the best performances among the evaluated serum inflammatory markers, their results should be interpreted with caution in clinical practice. Serum parameters may remain normal in chronic infections or may be elevated in patients with other inflammatory conditions. In addition, they also correlated poorly with synovial fluid leukocyte count and histology. Therefore, serum parameters are still insufficient to confirm or exclude a periprosthetic joint infection. Hence, they can only be recommended as suggestive criteria in diagnosing PJI

Background. Preoperative diagnosis of fracture related infections can be challenging, especially when confirmatory criteria such as sinus tract and purulent discharge are absent. Although serum parameters, such as CRP and white blood cell count (WBC), showed poor accuracy in the literature, they are still often used in clinical practice. The European Bone and Joint Infection Society (EBJIS) defined evidence-based criteria for fracture related infection. Elevated serum inflammatory markers were regarded as suggestive criteria only, as the literature was of limited quality. This study assessed the diagnostic value of the serum parameters CRP, WBC and differential cell count in the diagnosis of fracture related infections defined by the EBJIS-criteria for fracture related infections. Methods. In this retrospective cohort study, 94 patients who underwent surgical treatment for suspected infected non unions after failed fracture fixation were included. Preoperatively, blood samples including serum inflammatory markers were taken. For this study, cut-offs of 5 mg/L for CRP, 10×10⁁9 cells/L for WBC, and >70% for the percentage of neutrophils were regarded as positive for infection. All patients had intraoperative samples taken for microbiology and histology. Analysis of diagnostic accuracy was based on the receiver-operating characteristic (ROC). Results. Based on the EBJIS criteria, 40 patients (43%) were diagnosed with a fracture related infection. 11/94 (12%) patients had an elevated serum WBC count, 13/94 (14%) an increased percentage of neutrophils, and 43/82 (52%) an elevated serum CRP. The mean values of CRP concentration, WBC count, and percentage of neutrophils in the infection group were 7.9 mg/L (IQR:6.4 – 9.7), 18.3 G/l (IQR: 3.9 – 24.9), and 63% (IQR: 58 – 67%), respectively. The sensitivity, specificity, and area under the curve of serum WBC count were 20% (95% CI: 10 −35%), 94.4% (84 −99%), and 0.57 (0.50 – 0.64), respectively; of percentage of neutrophils 12.5% (5 – 27%), 85.2% (73 −93%), and 0.49 (0.42 – 0.56); and of serum CRP 67.6% (51 – 90%), 60.0% (45 – 73%), and 0.64 (0.53 – 0.74), respectively. A statistically significant difference between the AUCs of all three serum parameters and AUC of tissue culture as well as AUC of histology was shown (p <0.0001). A simple decision tree approach using only low WBC and CRP may allow identification of aseptic cases. Conclusion. Based on the standardized and evidence-based EBJIS criteria, the three inflammatory serum markers showed an insufficient accuracy for the diagnosis of fracture related infections. They also correlate poorly with culture or histological diagnosis. Therefore, they should not be used alone as a confirmatory test

Background. The diagnosis of periprosthetic joint infection (PJI) remains a challenge in clinical practice and the analysis of synovial fluid (SF) is a useful diagnostic tool. Recently, two synovial biomarkers (leukocyte esterase (LE) strip test, alpha-defensin (AD)) have been introduced into the MSIS (MusculoSkeletal Infection Society) algorithm for the diagnosis of PJI. AD, although promising with high sensitivity and specificity, remains expensive. Calprotectin is another protein released upon activation of articular neutrophils. The determination of calprotectin and joint CRP is feasible in a routine laboratory practice with low cost. Purpose. Our objective was to evaluate different synovial biomarkers (calprotectin, LE, CRP) for the diagnosis of PJI. Methods. In this monocentric study, we collected SF from hip, knee, ankle and shoulder joints of 42 patients who underwent revision or puncture for diagnostic purposes. Exclusion criteria included a joint surgery in the previous 3 months and a diagnosis of a systemic inflammatory disease. PJI was diagnosed in a multidisciplinary consultation meeting (RCP) of the Reference Centers for Osteoarticular Infections of the Great West (CRIOGO). SF was analysed for LE, CRP and calprotectin. The cut-off values used were 50 mg/L for calprotectin, 8.8 mg/L for CRP and 125 WBC/µL for LE. The overall sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were calculated for these different synovial markers. Results. Of the 42 patients included, 28 were considered as infected and 14 uninfected. The statistical parameters are presented in Table 1. Conclusion. The present study shows that the synovial calprotectin assay has an excellent sensitivity and a 100% NPV for the diagnosis of PJI, suggesting that a result < 50 mg/L could exclude PJI. This promising study suggests that calprotectin should be included with synovial CRP in a new decision algorithm for the diagnosis of PJI. For any tables or figures, please contact the authors directly

Hypochlorous acid (HOCl) is a potent anti-bacterial agent which could reduce periprosthetic joint infection. Early infection complications in joint replacements are often considered to be due to local contamination at the time of surgery and result in a significant socioeconomic cost. Current theatre cleaning procedures produce “clean” operating theatres which still contain bacteria (colony forming units, CFU). Reducing this bacterial load may reduce local contamination at the time of surgery. HOCl is produced naturally in the human neutrophil and has been implicated as the primary agent involved in bacterial killing during this process. In vitro research confirms its efficacy against essentially all clinically relevant bacteria. The recent advent of commercial production of HOCl, delivered as a fog, has resulted in extensive use in the food industry. Reported lack of corrosion and high anti-bacterial potency are seen as two key factors for the use of HOCl in the orthopaedic environment. Prior work by the authors comparing human cell toxicity of HOCl, chlorhexidine and iodine solutions shows favourable results. This study evaluates use of neutral HOCl applied as a dry room fog to decrease bacteria in the operating theatre environment. Using an animal operating theatre as the test site, bacterial swabs were taken from ten 100cm. 2. sample areas before standard cleaning with detergent, after standard cleaning, and again after 60 minutes exposure to HOCl fog. After standard cleaning, 6 of 10 sample sites recorded significant bacterial growth (>10 CFU/100cm. 2. ). After exposure to HOCl fog, growth in all 10 sites was below detection limits (<10 CFU/100cm. 2. ). This was repeated with specific exposure to Staphylococcus aureus and Escherichia coli. We can conclude that HOCl is effective when used as a fogging agent to reduce bacterial loading within an operating theatre environment and as such has significant potential to reduce intraoperative contamination and periprosthetic infection

Aim. Staphylococcus aureus (SA) can cause various infections and is associated with high morbidity and mortality rates of up to 40%. Antibiotic treatment often fails to eradicate SA infections even if the causative strain has been tested susceptible in vitro. The mechanisms leading to this persistence is still largely unknown. In our work, we to reveal SA interactions with host cells that allow SA to persist at the site of infection. Method. We established a sampling workflow to receive tissue samples from patients requiring surgical debridement due to SA bone-and joint or soft-tissue infections. We developed a multiplex immunofluorescent staining protocol which allowed us to stain for SA, leukocytes, neutrophils, macrophages, B-cells, T-cells, DAPI and cytoplasmatic marker on the same sample slide. Further, distance of SA to cell nuclei was measured. Interaction of immune cells and SA on a single cell level was investigated with high-resolution 3D microscopy. We then validated our findings applying fluorescence-activated cell sorting (FACS) on digested patient samples. Finally, we aimed to reproduce our ex vivo patient results in an in vitro co-culture model of primary macrophages and clinical SA strains, where we used live cell microscopy and high-resolution microscopy to visualize SA-immune cell interactions and a gentamicin protection assay to assess viability of SA. Results. Here, we revealed that CD68+ macrophages were the immune cells closest to SA with a mean distance of 56μm (SD=36.4μm). Counting the amount of SA, we found in total >7000 single SA in nine patients. Two-thirds of SA were located intracellularly. Two-thirds of the affected immune cells with intracellular SA were macrophages. The distribution of intra- to extracellular SA was independent of ongoing antibiotic therapy and underlying infection type. FACS confirmed these findings. In our co-culture model, intracellular SA remained alive for the whole observation period of eight hours and resided in RAB5+ early phagosomes. Conclusions. Our study suggests an essential role of intracellular survival in macrophages in SA infections. These findings may have major implication for future treatment strategies

Aim. To develop a new system for antibacterial coating of joint prosthesis and osteosynthesis material. The new coating system was designed to release gentamicin immediately after insertion to eradicate surgical contamination. Method. Steel implants (2×15mm) were coated with a solid nanocomposite xerogel made from silica and the dendritic polymer, hyperbranched polyethyleneimine. The xerogel was anchored inside a porous surface made by pre-coating with titanium microspheres. Finally, gentamicin was encapsulated in the xerogel, i.e. no chemical binding. A total of 50 µg gentamicin was captured into each implant. The efficacy of the new coating was evaluated in a porcine model of implant associated osteomyelitis. In total, 30 female pigs were randomized into 3 study groups (n=10). Group A; plain implants + saline, Group B; plain implants + 10. 4. CFU of Staphylococcus aureus, and Group C; coated implants + 10. 4. CFU of S. aureus. Implant + inoculum was placed into a pre-drilled implant cavity of the right tibia and the pig was euthanized 5 days afterwards. Postmortem microbiology and pathology were performed. Two additional pigs were used in a pharmacokinetic study where microdialysis (MD) catheters were placed alongside coated implants. Extracellular fluid was sampled regularly for 24 hours from the MD catheters and analyzed for gentamicin content. Results. Within Groups A and C, all implants were found sterile by sonication and bacteria could not be identified within the surrounding bone tissue. In contrast, all Group B animals had S. aureus positive implant and tissue microbiology. Macroscopic and microscopic pathological examinations confirmed that Group A and C animals were complete identic, i.e. no pus around implants and only minor peri-implant inflammation related to insertion of implants per se. All Group B animals had pus around their implants and a massive peri-implant inflammatory response dominated by neutrophil granulocytes. Maximum gentamicin release (35 µg /mL) was measured in the first obtained MD sample, i.e. after 30 min, and the concentration stayed above the MIC level for the used S. aureus strain for 8 hours. Conclusions. The new xerogel coating prevented development of osteomyelitis. Prevention was due to a fast gentamicin release immediately following insertion and antimicrobial active concentrations were detectable several hours after implantation. This means that the critical time point of most relevant surgical procedures potentially could be protected by the novel coating. The new coating will be investigated on larger scale implants and full-size prosthesis in the future

Aim. To investigate the local intra-operative concentration of gentamicin needed to prevent biofilm formation in a porcine model of implant-associated osteomyelitis. Method. In total 24 pigs were allocated to six groups. Group A (n=6) was inoculated with saline. Groups B (n=6), C (n=3), D (n=3), E (n=3) and F (n=4) were inoculated with 10 μL saline containing 10. 4. CFU of Staphylococcus aureus, however, different minimal inhibitory concentrations (MIC) of gentamicin were added to the inoculum of Groups C(160xMIC), D(1600xMIC), E(16000xMIC) and F(160000xMIC). The inoculums were injected into a pre-drilled implant cavity proximally in the right tibial bone. Following inoculation, a steel implant (2 × 15 mm) was placed in the cavity. The pigs were euthanized after five days. The implants were sonicated and swabs were taken from the implant cavity for microbiological evaluation. The peri-implant tissue was analyzed by histopathology including estimation of neutrophil infiltration. Results. The microbiological samples from Group A pigs were sterile. All implants and implant cavities of pigs inoculated with bacteria and bacteria + 160 or 1.600xMIC were positive for S. aureus. In each of the Groups E (16000xMIC) and F (160000xMIC) only one animal was found positive and 1/3 and 3/4 of the implants were sterile after sonication, respectively. All positive swabs were confirmed to be same spa-type as used for inoculation. By adding Groups C + D (<10000xMIC) and Groups E + F (>10000xMIC) a strong significant decrease (one-way ANOVA, P value = 0.001) of implant attached bacteria was only seen between the high MIC values and Group B (bacteria only). The histological examination demonstrated that 1600, 16000 and 160000 × MIC resulted in a peri-implant tissue reaction, including neutrophil estimation, comparable to saline inoculated animals. Patho-morphologically, it was not possible to distinguish between pigs inoculated with bacteria and bacteria + 160xMIC as both groups had a strong inflammatory response and an equal estimation of neutrophils. Discussion. The antibiotic susceptibility for prevention of an in vivo biofilm infection is influenced by body fluids, host immune response, extracellular host proteins like fibrin, tissue necrosis and development of an anaerobic environment. With the present in-vivo setup, we have demonstrated that local intra-operative gentamicin might be given in concentrations of more than 10000 times the MIC value in order to prevent biofilm formation by planktonic bacteria. Our study supports that biofilm susceptibility testing performed in-vitro is yet still unreliable for prediction of prophylactic and therapeutic success

Aim. Our goal is to assess diagnostic accuracy of synovial fluid testing in diagnosing prosthetic joint infection (PJI) as defined by the European Bone and Joint Infection Society (EBJIS). In addition to differential leukocyte count, simples and inexpensive biomarkers such as synovial fluid C-reactive protein (CRP), adenosine deaminase (ADA) and alpha-2-macrogloblulin(A2M) were also investigated and its possible role in increasing accuracy assessed. Method. Between January/2013 and December/2019 total hip or knee arthroplasty revision cases (regardless of preoperative diagnosis) were prospectively included provided enough synovial fluid for biomarker analysis was collected and at least four tissue samples, as well as the implant for sonication, were gathered for microbiological study. Definitive diagnosis was classified according to the new EBJIS PJI definition. Using receiver operating characteristic curves, we determined cutoff values as well as diagnostic accuracy for each marker. Results. Out of 364 revision arthroplasties performed, 102 fully respected inclusion criteria. There were 58 unlikely, 8 likely and 36 confirmed infections. Synovial fluid total leukocyte count, proportion of polymorphonuclear neutrophils (PMN), CRP, ADA and A2M were significantly different between groups. Area under the curve was 0.94 for total leucocyte count, 0.91 for proportion of PMN, 0.90 for CRP, 0.82 for ADA and 0.76 for A2M. Sensitivity, specificity, and predictive values for statistically optimal but also selected rule-in and rule-out cutoffs values are shown in Table 1. Interpreting a raised level of CRP(>2.7mg/L) or ADA(>60U/L) together with high leukocyte count (>1470 cells/μL) or proportion of PMN (>62.5%) significantly increases specificity and positive predictive value for affirming PJI. Conclusions. Differential leukocyte count cutoffs proposed by the EBJIS PJI definition are shown to perform well in ruling out (<1,500 cells/μL) and ruling in (>3,000 cells/μL) PJI. Adding simple and inexpensive biomarkers such synovial CRP or ADA is helpful in interpreting inconclusive results. For any tables or figures, please contact the authors directly

Introduction. Serum erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP), and synovial fluid white blood cell (WBC) count and differential are effective in diagnosing periprosthetic joint infection (PPJI); however their utility in patients with inflammatory arthritis is unknown. The purpose of this study is to determine the utility of these tests in patients with inflammatory arthritis. Methods. 934 Consecutive revision hip and knee arthroplasties were prospectively evaluated for PPJI. 202 Cases were excluded due to acute post-operative or hematogenous infection. 690 Patients had non-inflammatory and 42 had inflammatory arthritis. Receiver operating characteristic (ROC) curves were used to establish optimal ESR, CRP, WBC, and % neutrophil values for diagnosis of PPJI, and the area under the curve (AUC) was calculated to determine the overall accuracy. Results. The optimal thresholds for predicting PPJI were ESR 30mm/hr, CRP 17mg/L, WBC 2667, and differential 75% neutrophils in inflammatory arthritis, and ESR 32mm/hr, CRP 15mg/L, WBC 4000, and 78% neutrophils in non-inflammatory arthritis. The efficacy of these tests was similar in both populations (AUC for inflammatory ESR=86.2%, CRP=86.2%, WBC=93.8, 93.6% neutrophils; AUC for non-inflammatory ESR=85.2%, CRP=90.2%, WBC=94.5, 95% neutrophils); there was no significant difference between groups (ESR p = 0.861, CRP p= 0.549, WBC p=0.8315, % neutrophils p=0.7021). The rate of PPJI was significantly higher in patients with inflammatory (33.3%) than non-inflammatory (18.8%) arthritis (p-value=0.013). Conclusions. These results suggest that the ESR and CRP are useful in diagnosing PPJI in patients with inflammatory as well as non-inflammatory arthritis with similar optimal cut-off values

Objective. To investigate the effects of trauma and fracture surgery on leukocyte maturation and function. Background. Unbalanced inflammation triggered by trauma has been linked to multiorgan dysfunction (MOD) and death. In animal and cellular models, changes in neutrophil function and failure of monocyte infiltration and resolution have been implicated as possible causes. The investigators combine assays on neutrophil function with surface antigen expression on circulating neutrophils and monocytes. These are correlated with severity of traumatic injury, type of surgery and clinical outcome to help explain the aetiology of distant organ injury, and pose a case for damage control surgery. Results. A total of 20 patients requiring internal fixation of femoral shaft fractures, acetabular fractures and pelvic fractures were recruited. Those undergoing surgery following an interval period were used as control, with blood and plasma samples pre-operatively, and 2 and 5 days post-operatively, whilst patients with acute trauma also had an admission sample. Using flow cytometry, the neutrophils were gated on CD15+ CD14- with high side scatter whilst the monocytes were gated on CD14+ CD15- with low side scatter. Two days following surgery the neutrophils showed reduced CXCR2 expression and increased CXCR1, CD11b and IL-6R expression whilst the monocytes showed reduced CCR2 and HLA-DR receptor expression. The change in receptor expression was enhanced in the trauma patients in comparison to the control patients, and correlated with cellular function, using respiratory burst, elastase release and transmigration assays. Conclusions. This first human trial evaluating the immunologic/anti-inflammatory effects of trauma and trauma surgery on the specific antigen expression helps explain one mechanism for organ damage in the post-trauma patient

Aim. The cut-off values for synovial fluid leukocyte count and neutrophils differential (%PMN) for differentiating aseptic from septic failure in total knee arthroplasties were already defined in the past. Our goal was to determine the cut-off values for synovial fluid leukocyte count and %PMN in failed total hip arthroplasties (THA). Method. Patients undergoing revision THA were prospectively included. In perioperative assessment phase, synovial fluid leukocyte count and %PMN were determined. During the surgery, at least 4 intraoperative samples for microbiological and one for histopathological analysis were obtained. Infection was defined as presence of sinus tract, inflammation in histopathological samples, and ≥2 tissue and/or synovial fluid samples growing the same microorganism. Exclusion criteria were systemic inflammatory diseases, revision surgery performed less than 3 months from index surgery and insufficient tissue sampling. Receiver operating characteristic (ROC) curves were constructed to assess the diagnostic performance and Youden's J statistic was computed to identify optimal cut-off values. Results. During the study period (between June 2006 and June 2011) 227 revision THAs were performed by the senior author. 31 patients were excluded. 196 patients (mean age, 69 years; 68% females) with THA failure were included. Aseptic failure was diagnosed in 150 patients (76,5%) and THA infection was diagnosed in 46 patients (23,5%). Synovial fluid leukocyte counts were significantly higher in the infected group (median, 5.50×10. 6. leukocytes/ml range, 0.05 to 143.9×10. 6. leukocytes/mL) than in the aseptic group (median, 0.23×10. 6. cells/ml; range, 0 to 21.3×10. 6. leukocytes/ml, P<0,0001). The %PMN was also significantly higher in the infected group (median, 83%; range, 6% to 97%) than in the aseptic group (median, 27,5%; range, 0% to 94%, P<0,0001). A synovial fluid leukocyte count of > 1.54×10. 6. leukocytes/ml, had a sensitivity of 63%, specificity of 95%, positive and negative predictive values of 78% and 89%, respectively. A synovial fluid %PMN of > 64%, had a sensitivity of 65%, specificity of 93%, positive and negative predictive values of 73% and 90%, respectively. Conclusion. The synovial fluid leukocyte count of > 1.54×10. 6. leukocytes/ml and %PMN of > 64% are useful and reliable tests for excluding THA infection, having a negative predictive value of around 90%. This tests and calculated cut-off values are highly recommended in the diagnostic process of failed THAs

While advances in laboratory and imaging modalities facilitate the diagnosis of periprosthetic joint infection (PJI), clinical suspicion and a thorough history and physical remain the basis of evaluation. If clinical suspicion is high, the evaluation should be more vigorous, and vice versa. The erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) are inexpensive as well as ubiquitous, and should be obtained as a preliminary screening tool. These tests have been found to be cost-effective and highly sensitive. If both tests are negative, there is a low risk of periprosthetic joint infection (i.e., good negative predictive value). Positive results on both tests, in contrast, are not as specific but again raise suspicion. When either the ESR or CRP is elevated, or if the clinical suspicion for infection is high, aspiration of the knee joint is suggested. Synovial fluid should be sent for a synovial fluid white blood cell count (WBC), differential and culture. Given the ability to get three data points from one intervention, arthrocentesis, is the best single maneuver the physician can perform to rule in or out PJI. The synovial fluid WBC count has demonstrated in multiple studies excellent specificity and sensitivity in the diagnosis of infection. Based on multiple recent studies, the proceedings of the International Consensus on PJI recommend cut-offs for the synovial fluid WBC count as >3000 cells/mL and > 80% neutrophils for the differential. Synovial fluid biomarkers represent an expanding area of clinical interests based on the unique cascade of gene expression that occurs in white blood cells in response to pathogens. Deirmegian et al. described the unique gene expression and biomarker production by neutrophils in response to bacteria that are detectable in synovial fluid. Specifically, alpha-defensin is one such antimicrobial peptide. Along with synovial CRP, alpha-defensin can be measured in a currently commercially-available immunoassays. The diagnosis of PJI can be difficult to make in spite of the variety of tests available. That being said, the diagnosis is easily made in our experience in 90% of patients by getting an ESR and CRP followed by selective aspiration of the joint if these values are elevated or if the clinical suspicion is high. Synovial fluid obtained should be sent for a synovial fluid WBC count, differential and cultures

The diagnosis of prosthetic-joint infection (PJI) is challenging, as bacteria adhere on implant and form biofilm. Therefore, current diagnostic methods, such as preoperative culture of joint aspirate have limited sensitivity with false-negative results. Aim. To evaluate the performance of measurement synovial fluid (SF) D-lactate (as a pathogen-specific marker) for the diagnosis of PJI and estimate of treatment success. Method. 224 patients undergoing removal knee or hip prosthesis were included in the study between January 2015 and March 2017. 173 patients of this group had aseptic loosening of prosthesis and 87 were diagnosed with PJI. Prior to surgery, synovial fluid routine culture, D-lactate test, leukocyte count and neutrophils (%) were performed for each patient. In order to evaluate a treatment success, the measurement of SF D-lactate before second two-stage exchange procedure (after treatment) was implemented in 30 patients. Diagnosis of PJI was established according to modified Zimmerli criteria. Results. Of 87 patients with infection of prosthetic joints, 61 (70%) had positive synovial fluid cultures, including Staphylococcus spp. (70%), Streptococcus spp. (10%), Enterococcus spp. (6%), Anaerobes (6%), Enterobacteriacae (4%), P. aeruginosa (2%), C. parapsilosis (2%). There was no significant difference in SF D-lactate levels due to different bacterial strains. The optimal D-lactate cut off was 1,2 mmol/l (sensitivity = 98%, specificity = 84%, PPV = 79%, NPV = 98%, AUC 0,99). Concentration of SF D-lactate was significantly higher in patients with PJI compared to aseptic loosening of prosthesis (median (range)) 2.33 (0.99–3.36) vs 0.77 (0.01–2.4), p<0.0001.D-lactate has better sensitivity for diagnosis of PJI (98%), compared to leukocytes (80%) and neutrophils % (89%), p<0.0001). The concentration of D-lactate decreased below cut off within four weeks after revision surgery (after treatment) in all patients except of three, showing relapse of infection (p<0.0001). Conclusions. The measurement of synovial fluid D-lactate demonstrated high analytical performance in the diagnosis of PJI, it is a reliable pathogen specific marker. D-lactate has the best sensitivity as independent diagnostic method and could be implemented for the evaluation of treatment success

Introduction. In the last couple of years, several synovial biomarkers have been introduced in the diagnostic algorithm for a prosthetic joint infection (PJI). Alpha defensin-1 proved to be one of the most promising, with a high sensitivity and specificity. However, a major disadvantage of this biomarker is the high costs. Calprotectin is a protein that is present in the cytoplasm of neutrophils, and is released upon neutrophil activation. Its value has been established for decades as a (fecal) marker for inflammatory bowel disease. Aim. To determine the efficacy of synovial calprotectin in the diagnosis of a prosthetic joint infection. Methods. We prospectively collected synovial fluid (from hip, knee and shoulder) from patients with a proven PJI (n=15) and from patients that underwent a revision surgery without signs of a PJI (n=19). Patients with an active rheumatoid arthritis and/or gout were excluded from the study. Synovial fluid was centrifuged and the supernatant was used to measure calprotectin, by using a rapid, point of care test. This test was validated for synovial fluid analysis of calprotectin using an ELISA. A Mann-Whitney U test was used to calculate the difference between both patient groups. Results. The median calprotectin level was 899 mg/L (range 28–2120) for PJI versus 22 mg/L (range 0–202) for controls (p < 0.0001). With a cut-off value of 50 mg/L, synovial calprotectin has a high sensitivity of 93%, and a specificity of 84%. The positive and negative predictive values are 82% and 94%, respectively. Conclusions. Synovial calprotectin is a potentially valuable biomarker in the diagnosis of a PJI. With a point of care test, a rapid quantative diagnosis can be made within the operating room (results are obtained within 20 minutes), and could help in the decision making process to re-implant a prosthesis in a one stage procedure. In comparison to the currently available test (to measure alpha defensin-1), the measurement of calprotectin test is much cheaper (500 euro versus 20 euro per sample) and easily to implement in hospitals where this test is already available. Its diagnostic efficacy for exclusively low-grade PJI should be further elaborated

Aim. When a prosthetic joint infection (PJI) is suspected, guidelines recommend performing periprosthetic samples, at least one for histopathological examination and 3 to 6 for microbiological culture. The diagnosis of infection is based on the presence of neutrophil granulocytes whose number and morphology can be variable, resulting in definition of “acute” inflammation. The acute inflammation of periprosthetic tissue is supportive of infection. Since 2007, in our hospital, for all patients with suspected PJI who underwent surgery, from each sample taken by the surgeon, one part has been sent to the pathologist and the other one to the microbiologist. Our aim was to compare histopathological to microbiological results from samples taken intraoperatively at the same site. Method. We conducted a retrospective study including all surgeries for which at least one couple “histopathology-culture” was found. Exclusion criterion was a history of antimicrobial treatment 2 weeks prior the surgery. Results. From July 2007 to April 2015, 309 surgeries for suspected PJI were performed in 181 patients. Median age of the study population was 70 years, 60% of patients were male, 45% had a history of joint infection. The location of arthroplasty was knee in 50% of cases and hip in 46%, ankle and shoulder in 4%. Surgery was performed within one month after the last prosthetic surgery in 15% of cases. According to the criteria from the Musculoskeletal Infection Society, 60% of cases should have been considered as having an infection. The median number of samples per surgery was 4 (IQR 3–5) for histopathological examination and 5 (IQR 4–6) for culture. Finally, 1247 couples “histopathology-culture” were available. Among them, histopathological examination showed acute inflammation in 292 cases (23%) and subacute inflammation in 327 cases (26%). Microorganisms considered to be pathogenic were found in 582 samples (47%). The presence of neutrophil granulocytes was well correlated with the presence of those microorganisms (OR=4.1; IC 95% 3.1–5.5). As expected, the highest correlation between acute inflammation and positive culture was observed for early infection (< 1 month) (OR = 9; 3.6–23.4) and Staphylococcus aureus infection (OR = 4.8; 3.3–7.0). There was no correlation between acute or low-grade inflammation and anaerobic or Candida infection. Conclusions. Our results confirmed histopathological examination is better correlated with culture in acute infection and/or infection due to highly virulent bacteria but must be interpreted with caution in case of chronic infection or infections due to microorganisms with low virulence

To test the hypothesis that: CERAMENT[™]|G (C-G) would improve new bone growth and decrease infection rate after debridement as compared with 1) CERAMENT|BONE VOID FILLER (CBVF) and 2) no void filler in a rat osteomyelitis model. 72 Sprague Dawley rats were injected with 1.5 × 10∧6 CFU of S. aureus into a drill hole in the right tibia. After 3 weeks, the osteomyelitic defect was debrided, and filled with either: 1) C-G (n=32), 2) CBVF (n=20), or 3) nothing (n=20). 6 weeks after the second surgery, 20 rats from each group were sacrificed and the right tibias were harvested. A long-term group (n=12) of C-G treated rats were also sacrificed at 6 months after the second surgery. The tissues were sonicated and the colony forming units in the sonicate were quantified by serial dilutions and culture. MicroCT was used to quantify the new bone growth (BV/TV) in the debrided osteomyelitic void. Histological samples were analyzed for the presence of a neutrophil response by a blinded pathologist. (*: p<0.05). Positive cultures in:. ○ 30% of animals treated with CBVF. ○ 25% of animals treated with no void filler. ○ 0% of animals treated with C-G (*). Neutrophil reaction in:. ○ 35% of animals treated with CBVF. ○ 50% of animals treated with no void filler. ○ 0% of animals treated with C-G (*). The BV/TV in:. ○ C-G treated rats was 24% greater than CBVF treated rats (*). ○ C-G treated rats was 94% greater than rats treated with no void filler (*). ○ CBVF treated rats was 56% greater than rats treated with no void filler (*). Animals sacrificed at 6 months which were treated with C-G did not have any evidence of infection by culture or histology. The bone mass of the implanted limb was higher than the contralateral (non-operated) side. CERAMENT|G decreased the rate of infection and increased new bone growth as compared with both CBVF and no void filler in a debrided osteomyelitic environment. Animals treated with C-G at 6 months showed no evidence of infection and retained a higher bone mass relative to the contralateral (non-operated) side. This study supports the use of CERAMENT|G as a readily available void filler which could be used in osteomyelitic environments after debridement