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

Aim. The aim of the study is to evaluate the specificity and sensibility of leukocyte esterase for the diagnosis of periposthetic joint infection (PJI). Method. Between October 2016 and April 2017 we enrolled 65 patients underwent to hip and knee revision arthroplasty due to uncertain joint infection. Synovial fluid was obtained from 64 joints that underwent revision arthroplasty. Each patient was evaluated in the preoperative time with CRP, ESR and leukoscan, in the intraoperative time with frozen section and leukocyte esterase strip and post-operative with sonication fluid culture, periprosthetic tissues cultures and histological examination. Results of all of these exams were compared to assess the specificity, the sensibility, the positive and negative predicting values of leukocyte esterase for the diagnosis of PJI. Results. The leukocyte esterase test with a threshold of +/++ had a sensitivity of 80.2%, a specificity of 82.8%, a positive predictive value of 63.8%, and negative predictive value of 92.1%. Using the threshold of ++ as a positive leukocyte esterase result, the specificity reached 97.8%, the positive predictive value 90.8%, and the negative predictive value 89.0%. Conclusions. These results demonstrate that leukocyte esterase is a quite accurate, effective marker of periprosthetic joint infection and that it is a valuable tool that can be used in conjunction with the the other tests for diagnosis of PJI

Background. Periprostetic joint infections (PJI) are often difficult to diagnose, to treat and often leave the patient with severe impaired function. The presence of low virulent bacteria is frequently discovered in apparent aseptic revisions of shoulder arthroplasties and pose a challenge to diagnose preoperatively. Dual Isotope In111 Leucocyte/ Tc99 Bone Marrow SPECT CT scan (L/BMS) is considered the radionuclide gold standard in preoperative diagnosing PJI with reported high specificity and sensitivity in hip and knee arthroplasties. Unfortunately, it is labour-intensive and expensive to perform and documentation using L/BMS on shoulder arthroplasties lack. Aim. To investigate if L/BMS succeeds in detecting shoulder PJI compared to tissue cultures obtained perioperatively. Method. All patients referred to a highly-specialised shoulder department with a painful or stiff shoulder-arthroplasty were included in the cohort. To diagnose infection as a possible cause of arthroplasty failure a L/BMS was planned for all patients. If the arthroplasty was revised, 5 tissue biopsies were obtained from the most infection-suspicious site during revision. Biopsies were cultured in broth and on plates for 14 days due to the high frequency of low virulent infection in shoulder revisions. Infection was defined as growth of the same bacteria in 3 or more of 5 the biopsies. Results. During the observation period 71 patients were referred. Revision surgery was performed in 62% of the patients (44/71) of which 29 also had been examined by L/BMS. A microbiological diagnose was available for all. The most predominant organism isolated was P. Acnes. Two patients both had a positive L/BMS and positive cultures. Negative L/BMS and negative cultures were found in 20 patients. The remaining 7 patients had negative L/BMS, but positive cultures. The two patients with a positive L/BMS both showed overt clinical signs of infection. L/BMS show a sensitivity 0.22 95%CI(0–0.49) and specificity 1.00 95%CI(1.00–1.00) in detecting shoulder PJI. The Positive Predictive Value is 1.00 95%CI(1.00–1.00) and Negative Predictive Value 0.74 95%CI(0.57–0.90). No patients infected with P. Acnes resulted in a positive scintigraphy nor had they preoperative or perioperative signs of infection. Conclusion. Only patients with severe infectious symptoms of shoulder PJI resulted in positive L/BMS. Hence, the scan added nothing to the preoperative clinical diagnose. In111 Leucocyte/ Tc99 Bone Marrow SPECT CT scan cannot be recommended as a standard screening procedure when evaluating failed shoulder arthroplasties for possible infection

Aim. Treatment of complicated wound healing after total joint arthroplasty is controversial. What exactly constitutes prolonged wound drainage is matter of debate and recommendations to manage it vary considerably. Nonoperative measures are often recommended. If drainage persists, surgery may be indicated. To further intricate decision-making, differentiating superficial from deep surgical site infection is also controversial and inherently complex. Specific cutoffs for synovial fluid leukocyte count and blood C-reactive protein (CRP) in the acute stage have been suggested as a way to superficial infection requiring superficial wound washout from deep infection requiring a formal debridement, antibiotics and implant retention (DAIR) procedure. The goal of this study is to analyze clinical and laboratory findings of an institutional protocol of “aggressively” proceeding with formal DAIR in all patients with complicated wound healing. Method. Our indications for DAIR in suspected acute postoperative periprosthetic joint infection (PJI) are: 1)prolonged wound drainage and CRP upward trend after day-3; 2)persistent wound drainage by day-10 regardless of CRP; 3)wound healing disturbance (e.g. “superficial” infection, “superficial” skin necrosis) anytime in early postoperative weeks. We retrospectively evaluated patients undergoing DAIR in the first 60 postoperative days between 2014–2018. Patients without multiple deep tissue cultures obtained intraoperative were excluded. Deep infection was defined by at least two positive deep tissue cultures or one positive deep culture and positive leukocyte count (>10,000 cells/mL or >90% PMN). Results. A total of 44 DAIR procedures were included. Deep infection was confirmed in 79.5%(35/44) of cases. Mean CRP in infected cases was 93mg/L with 63%(19/30) of them below the 100 mg/L threshold. Unfortunately, only a small proportion of cases (10/44) had synovial fluid leukocyte counts available. Mean leukocyte count was 15,558 cells/mL and mean proportion of PMN was 65.3%. Of these ten, six confirmed deep infections were below the proposed >10,000 cells/mL or >90% PMN cutoff. Conclusions. Early diagnosis of acute postoperative PJI is often hampered by its very subtle presentation. This study confirms that more often than not, deep infection is present when facing complicated wound healing after total joint arthroplasty, supporting our institutional “aggressive” protocol. We have been surprised by the number of confirmed acute PJI with low blood CRP levels and low synovial leukocyte counts. We hypothesize that the proposed acute PJI specific thresholds may lead to misinterpret a significant proportion of cases as superficial infections thus compromising timely intervention. The findings of this study lack confirmation in larger cohorts

Culture examination is still considered the gold standard for diagnosis of bone and joint infections, including prosthetic ones, even if in up to 20–30% of cases, particularly prosthetic joint infections, it fails to yield microbial growth. To overcome this limitation, determination of markers of inflammation and or infection directly in joint fluid has been proposed. Aim of this study was to evaluate the applicability of measurement of lecukocyte esterase (LE), C-reactive protein (CRP) and glucose in synovial fluid for diagnosis of bone and joint infections. Synovial fluids from 80 patients were aseptically collected and sent to laboratory for microbiological cultures. After centrifugation at 3000 rpm for 10 minutes, pellet was used for cultures, while the surnatant was used for determination of LE, CRP and glucose. LE and glucose were evaluated by means of enzymatic colorimetric strips developed for urinanalysis. One drop of synovial fluid was placed on the LE and on the glucose pads and the results were read after about 120 seconds. A LE test graded + or ++, and a glucose test equal to trace or negative were considered suggestive for infection. CRP was measured by an automated turbidimetric method. On the basis of clinical findings, microbiological, haematological and histological analyses patients were retrospectively divided into 2 groups. Group 1 comprised 19 infected patients (12 males, 7 females age: 70.6 ± 10.3 yrs, range: 47 – 88 yrs) while Group 2 included 61 aseptic patients (32 males and 29 females, age: 61.5 ± 16.3 yrs, range: 15 – 84). Sensitivity of the three tests was 89.5%. 84% and 73,7% for LE, CRP and glucose, respectively. Specificity was 98.4%, 88.5% and 70% for LE, CRP and glucose, respectively. Positive and negative predictive values were 94.4% and 96.8% for LE, 69.6% and 94.6% for CRP and 77.8% and 89.6% for glucose test. When LE was combined with CRP, sensitivity increased to 94.7%, while no differences were observed for LE combined with glucose. Leukocyte esterase has proven to be a rapid, simple and inexpensive test to rule in or out bone and joint infections. Combination of its measurement with that of CRP increased sensitivity. In conclusion, the combination of leukocyte esterase and CRP may represent a simple and useful tool for diagnosis of bone and joint infections

Injection before total knee arthroplasty(TKA) is the one of the postoprative risk factors after TKA and Infection after TKA can result in disastrous consequences. When the duration between injection and TKA is longer than 6 months, the risk is no longer elevated. Evaluation of synovial WBC number in frozen section slide is needed to check the presence of infection in revision total knee arthroplasty. Currently many patients have a history of multiple intraarticular injection before the primary TKA. Purpose of this study is to evaluate the synovial WBC findings in primary TKA and compare between injection group and no injection group. Materials and Methods. The synovial specimen(suprapatella pouch and posterior capsule) of 68 primary total knee arthroplasty were evaluated by the pathologist and reported the number of the WBC in frozen section /5 separate high power fields(HPF) (500x).. Injection group were 37 cases and non -injection group were 31 cases. Preoperative CRP and ESR were recorded and followe-up duration was more than 2 years. Joint fluid was sent to be cultured and analysed. Results. WBC count in frozen section shoed was average 4 WBCs/HPF (range < 0∼ 25) in both specimen and the suprapatella specimen was 3 WBCs/HPW (range 0∼25) and posterior capsule specimen was 1 WBCs/HPF(range 0∼14). The WBC count of injection group was 8 (range, 0∼25) and that of no injection group was 1.2 cells (range 0∼12) (p<0.05). The WBC counts in joint fluid was average 240 cells/ml (range. 1∼300) in non injection group and 643 cells/ml(range, 50∼1000) (p<0.05). The duration from the intraarticular injection to index surgery was 9 months(range, 6 weeks∼ 7 momths). The number of injection and duration bwtween injection and operationto has no significant correlation with the WBC counts. Eight percentage of specimen showed more than 10 WBCs in injection group and these patients have been not infected after more than 24 moths after TKA. Conclusion. The WBC count of the synovium in priamry TKA with injection history for degenerative osteoarthritis is variable and we could not recommend the routine frozen section analysis in primary TKA who have a history of intraarticualr injection..

Aim

The purpose of this work is to study whether there is or not, in the case of an aseptic arthroplasty exchange, a relationship between positive cultures and an early periprosthetic joint infection.

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.

Aim. Synovial fluid D-lactate may be useful for diagnosing septic arthritis (SA) as this biomarker is almost exclusively produced by bacteria. We evaluated the performance of synovial fluid D-lactate and determined its optimal cut-off value for diagnosing SA. Method. Consecutive patients with suspicion of septic arthritis were prospectively included. They underwent joint aspiration and synovial fluid was collected for culture, leukocyte count and D-lactate concentration (by spectrophotometry). Youden's J statistic was used for determining optimal D-lactate cut-off value on the receiver operating characteristic (ROC) curve by maximizing sensitivity and specificity. Results. A total of 155 patients were included. Using institutional criteria, 21 patients (14%) were diagnosed with SA and 134 (86%) patients with aseptic arthropathy, out of which 43 (27%) had osteoarthrosis, 80 (52%) had rheumatic arthropathy and 11 (7%) reactive arthritis. The optimal cut-off of synovial fluid D-lactate to differentiate SA from aseptic cases was 0,035 mmol/l. Synovial fluid D-lactate had a sensitivity 90% (95% CI: 70–99%) and specificity 87% (95% CI: 80–92%) compared to leukocyte count with sensitivity 81% (95% CI: 60–95%) and specificity 83% (95% CI: 76–90%). Culture was positive in only 17 (80%) out of 21 patients with SA. Conclusions. The synovial fluid D-lactate showed high sensitivity and specificity for diagnosis of SA which was higher than the current gold standard of diagnosis (culture and leukocyte count). The high sensitivity makes this biomarker useful as a point-of-care screening test for SA

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

Aim. The analysis of synovial fluid has proved to be of crucial importance in the diagnostic process of prosthetic joint infections (PJI), suggesting the presence of an infection before the microbiological culture results. In this context, several studies illustrated the efficacy of synovial calprotectin in supporting the diagnosis of PJI [1, 2]. However, several testing methods have been explored to detect synovial calprotectin levels, emphasizing the need to use a standardized, rapid and rapid test. In this study, synovial calprotectin was analyzed by means of a commercial stool test [3] to explore whether the detected levels might predict PJIs and, therefore, being a promising tool for the fast and reliable diagnosis of this complication. Method. The synovial fluid of 55 patients underwent to revision of the prosthetic implant were analyzed. The measurement of calprotectin was carried out by of commercial stool test, following the protocol for liquid samples. Calprotectin levels were then compared to other synovial biomarkers of PJI such as leucocyte esterase and count and percentage of polymorphonuclear cells. Data analysis were performed using R software v4.1.1 (R Core Team) and package “pROC” [4]. Receiver operator characteristics curves were designed using culture test as gold standard to evaluate the area under curve (AUC) of each method (with DeLong method for confidence-interval calculation). Thresholds were calculated to maximize Youden's index; sensitivity and specificity were reported. One-to-one Pearson's correlations coefficient were calculated for each pair of methods. P value <0.05 were considered statistically significant. Results. Of the 55 synovial fluids analyzed, 13 patients were diagnosed with PJI and 42 with an aseptic failure of the implant. The specificity, sensitivity, and AUC of calprotectin resulted 0.90, 0.85, and 0.86 (95%CI: 0.72–0.99), respectively with a set threshold of 226.5 µg/g. The values of calprotectin had a moderate and statistically relevant correlation with the synovial leucocyte counts (r. s. = 0.54, p = 0.0003) and the percentage of polymorphonuclear cells (r. s. = 0.68, p = 0.0000). Conclusions. From this analysis, it can be concluded that synovial calprotectin is a valuable biomarker that correlates with other established indicator of local infection, delivering a rapid and reliable results and supporting the diagnostic process of PJI

Aim. Septic arthritis (SA) is considered a medical emergency. The most common etiological agents are glucose consuming bacteria, so we evaluated the clinical utility of synovial fluid (SF) glucose levels and other biochemical parameters for supporting the diagnosis of the disease and their association with a positive bacteria culture and joint destruction. Methods. Adult patients with SA diagnose were enrolled prospectively between July 2018 and October 2019. As control group, adults with knee osteoarthritis, meniscus and/or knee ligaments lesions were enrolled. SF samples were obtained from the joints by arthrocentesis/arthrotomy. Microbiological analyses of SF were performed using Brucella broth blood culture flasks, samples were incubated at 37°C with 5% CO. 2. for 24 hours. Gram stain, chocolate and blood agar were used for the identification and growth of the bacteria. SF glucose levels, pH and leukocyte esterase were measured as biochemical parameters using a glucometer and colorimetric test strips. The Outerbridge classification was used for grading the osteochondral injury. Furthermore, blood samples were collected from patients and control subjects for determining glucose levels. Results. We included 8 subjects with knee ligaments lesions, 6 with meniscus lesions and 5 with osteoarthritis as control group, as well as 20 patients with SA diagnose. The mean age of the patients was 57.8 years with a 65% of male predominance. The most common affected joint was the knee (85%). SF culture was positive in 60% of the cases and the most common etiological agent was Staphylococcus aureus (58.3%). SF glucose levels from patients were lower than the controls (P=0.0018) and showed the lowest concentration in patients with a positive culture (P=0.0004). There was also a difference between blood and SF glucose concentration from the positive culture patients (P<0.0001). Leucocyte esterase presented the highest values in positive culture patients (P=<0.0001) and a more acidic pH was found compared to the control group (P<0.0001). Regarding the osteochondral injury, the lowest concentrations of SF glucose were found in patients with a higher grade in the classification (P = 0.0046). Conclusions. SF glucose and leukocyte esterase concentrations might be a quick and cheap useful parameter for the physician for distinguishing between bacterial infection and not infected joint. In addition, the lowest SF glucose levels might give information about the joint damage due to the disease

Aim. Accurate diagnosis is key in correctly managing prosthetic joint infection (PJI). Our aim is to compare the preoperative performance of three PJI definitions comparing it to definitive postoperative classification. Method. This is a multicenter retrospective study of patients who have undergone total hip or knee revision surgery in four different European institutions. For this specific study, cases with no preoperative synovial fluid differential leukocyte count and less than four intraoperative microbiology samples were excluded. Cases were classified using the 2021 EBJIS, the 2018 International Consensus Meeting (ICM) and the 2013 Musculoskeletal Infection Society (MSIS) PJI definitions. Preoperative classification was based on clinical features, inflammatory markers and synovial fluid leukocyte count and microbiology results. Results. Preoperative and definitive PJI classification status of the 384 patients included are presented in figure 1. EBJIS definition showed the highest agreement between preoperative and definitive classification (k=0.86, CI95% 0.81–0.90, p<0.001) compared to ICM 2018 (k=0.80, CI95% 0.75–0.84, p<0.001) or MSIS 2013 (k=0.70, CI95% 0.62–0.77, p<0.001). Compared to its respective definitive classification: EBJIS preoperative unlikely result shows 86.8% (95%CI 81.3%–91.2%) sensitivity and 87.7% (95%CI 83.3%–91.1%) negative predictive value (NPV); ICM 2018 preoperative not infected result shows 83.5% (95%CI 77.4%–88.5%) sensitivity and 86.2% (95%CI 81.9%–88.6%) NPV and; MSIS 2013 preoperative not infected result shows 63.9% (95%CI 55.0%–72.1%) sensitivity and 84.3% (95%CI 81.1%–87.1%) NPV. Around half of the preoperative EBJIS likely (45.8%) and ICM 2018 inconclusive (54.5%) turn out to be infected postoperatively. If we consider the more sensitive definition (EBJIS) as the gold standard: ICM 2018 preoperative not infected result shows 75.1% (95%CI 68.5%–81.0%) sensitivity and 78.3% (95%CI 73.9%–82.2%) NPV and; MSIS 2013 preoperative not infected result shows 42.1% (95% CI 35.2%–49.4%) sensitivity and 62.0% (59.2%–64.8) NPV. Conclusions. The EBJIS 2021 definition is not only the most sensitive definition as it was shown to be the most effective in preoperatively ruling out PJI when there is a negative result. For any tables or figures, please contact the authors directly

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

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 evaluate the analytical performance of synovial fluid D-lactate test for the diagnosis of PJI. Method. Consecutive patients undergoing diagnostic joint aspiration of prosthetic joint were prospectively included. PJI was diagnosed according to the proposed European Bone and Joint Infection Society (EBJIS) definition criteria. Synovial fluid was collected for culture, D-lactate measurement (by spectrophotometry, λ = 570 nm) and leukocyte count and differential (by flow cytometry). The receiver operating characteristic (ROC) analysis was performed to assess the diagnostic performance of D-lactate and leukocyte count. Results. Diagnostic joint aspiration was performed in 224 patients with prosthetic joints. PJI was diagnosed in 87 patients (39%). The optimal D-lactate cut-off value for diagnosing PJI was 1.2 mmol/l. The sensitivity of synovial fluid D-lactate was 97.7%, specificity 83.9%, whereas the sensitivity of synovial fluid leukocyte count was 87.5% with specificity 95.7%. 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. We found positive correlation between D-lactate and erythrocytes in synovial fluid sample in the aseptic group (ρ = 0.339, p< 0.01). Conclusions. The synovial fluid D-lactate showed a good diagnostic performance for the diagnosis of PJI, which was comparable to the synovial fluid leukocyte count. Currently available (UV)-based method for detection of D-lactate showed low specificity (84%) due to influence of hemoglobin with the similar absorbance wavelengths (λ = 540 nm). More specific high-performance methods such as electro-chemical sensing system or lateral flow immunochromatographic assays should be implemented

Aim. Differentiation of infected (INF) nonunion from aseptic (AS) nonunion is crucial for the choice of intra- and postoperative treatment. Preoperative diagnosis of infected nonunion is challenging, especially in case of low-grade infection lacking clinical signs of infection. Standard blood markers such as C-reactive protein or leucocyte count do not aid in preoperative diagnosis. Proteomic profiling has shown promising results for differentiation of numerous chronic disease states, and in this study was applied to preoperative blood samples of patients with nonunion in an attempt to identify potential biomarkers. Method. This prospective multicenter study enrolled patients undergoing revision surgery of femur or tibia nonunion. Patients with implant removal after regular fracture healing (HEAL) were included as a control-group. Preoperative blood samples, intraoperative tissue samples, sonication of osteosynthesis material and 1-year-follow-up questionnaire were taken. Nonunion patients were grouped into INF or AS after assessing bacterial culture and histopathology of retrieved samples. Diagnosis of infection followed the fracture related infection consensus group criteria, with additional consideration of healing one year after revision surgery. Targeted proteomics was used to investigate a predefined panel of 45 cytokines in preoperative blood samples. Statistical differences were calculated with Kruskal Wallis and Dunn's post hoc test. Cytokines with less than 80% of samples being above the lower limit of detection range (LLDR) were excluded for this study. Results. We recruited 62 AS, 43 INF and 32 HEAL patients. Patients in the two nonunion groups (INF and AS) did not differ concerning smoking, diabetes or initial open or closed fracture. Thirty-two cytokines were above LLDR in >80% of patients. INF patients showed a significant difference in expression of 8 cytokines compared to AS, with greatest differences observed for Macrophage Colony Stimulating Factor 1 (MCSF-1) and Hepatocyte Growth Factor (HGF) (p<0.01). In comparing AS with HEAL patients, 9 cytokines displayed significant differences, including interleukin (IL)-6, Vascular Endothelial Growth Factor A (VEGFA), Matrix Metalloproteinase 1 (MMP-1). Comparison of INF with HEAL patients revealed significantly different expression of 20 cytokines, including. IL-6, IL-18, VEGFA or MMP-1. Conclusions. Our study revealed differences in plasma cytokine profile of blood samples from INF and AS patients. Although no single biomarker is sufficient to differentiate these patients preoperatively in isolation, future multivariant analysis of this cytokine data in combination with clinical characteristics may provide valuable diagnostic insights. Funded by German Social Accident Insurance (FF-FR 0276) and AO Trauma (AR2021_04)

Aim. The most frequent mechanical failure in the osteosynthesis of intertrochanteric fractures is the cut-out. Fracture pattern, reduction quality, tip-apex distance or the position of the cervico-cephalic screw are some of the factors that have been associated with higher cut-out rates. To date, it has not been established whether underlying bacterial colonization or concomitant infection may be the cause of osteosynthesis failure in proximal femur fractures (PFF). The primary objective of this study is to assess the incidence of infection in patients with cut-out after PFF osteosynthesis. Method. Retrospective cohort study on patients with cut-out after PFF osteosynthesis with endomedullary nail, from January 2007 to December 2020. Demographic data of patients (such as sex, age, ASA), fracture characteristics (pattern, laterality, causal mechanism) and initial surgery parameters were collected (time from fall to intervention, duration of surgery, intraoperative complications). Radiographic parameters were also analyzed (tip-apex distance and Chang criteria). In all cut-out cases, 5 microbiological cultures and 1 anatomopathological sample were taken and the osteosynthesis material was sent for sonication. Fracture-related infection (FRI) was diagnosed based on Metsemakers et al (2018) and McNally et al (2020) diagnostic criteria. Results. Of the 67 cut-out cases, 16 (23.9%) presented clinical, analytical or microbiological criteria of infection. Of these sixteen patients, only in 3 of them the presence of an underlying infection was suspected preoperatively. A new osteosynthesis was performed in 24 cases (35.8%) and a conversion to arthroplasty in the remaining 43 (64.2%). A comparative analysis was performed between cases with and without infection. The groups were comparable in terms of demographic data and postoperative radiological data (using Chang criteria and tip-apex distance). Patients with underlying infection had a higher rate of surgical wound complication (56.3% vs 22%, p = 0.014), higher rates of leukocytes counts (11.560 vs 7.890, p = 0.023) and time to surgery (5.88 vs 3.88 days, p = 0.072). Conclusions. One out of four osteosynthesis failure in PFF is due to underlying FRI and in almost 20% were not unsuspected before surgery. In PFF osteosynthesis failures, underlying infection should be taken into account as a possible etiological factor and thus a preoperative and intraoperative infection study should be always performed

Aim. The aim of the study was to assess the accuracy of the alpha defensin lateral flow test for diagnosis of periprosthetic joint infection (PJI) using an optimized diagnostic algorithm and three classification systems. In addition, we compared the performance with synovial fluid leukocyte count, the most sensitive preoperative test. Method. In this prospective multicenter study we included all consecutive patients with painful prosthetic hip and knee joints undergoing diagnostic joint aspiration. Alpha defensin lateral flow test was used according to manufacturer instructions. The following diagnostic criteria were used to confirm infection: Musculoskeletal Infection Society (MSIS), Infectious Diseases Society of America (IDSA) and Swiss orthopedics and Swiss Society of Infectious Diseases (SOSSID). In the latter, PJI was confirmed when at least one of following criteria applied: macroscopic purulence, sinus tract, positive cytology of joint aspirate (>2000 leukocytes/μl or >70% granulocytes), histological proof of acute inflammation in periprosthetic tissue, positive culture (from aspirate, tissue or sonication fluid). Infection was classified as chronic, if symptom duration was more than 3 weeks or if infection manifested after more than 1 month after surgery. The sensitivity and specificity of the alpha defensin lateral flow test and leukocyte count in synovial fluid were calculated and compared using McNemar Chi-square test. Results. Of 151 included patients evaluated for painful prosthetic joints (103 involved knees, 48 hips), the median patient age was 69 years (range, 41–94 years) and 75 patients were female. Systematically evaluating the included patients according to the different diagnostic criteria, MSIS and IDSA revealed both 33 patients with PJI (22%), whereas SOSSID disclosed 47 septic failures (31%), among them 36 chronic infections (77%). Sensitivity of the test was 79% when applying MSIS criteria, 70% with IDSA criteria and 57% with SOSSID criteria. Specificity ranged from 96% (IDSA) to 98% (MSIS) and 99% (SOSSID). Applying the most stringent definition criteria (SOSSID), leukocyte count showed significantly higher sensitivity than the alpha defensin lateral flow test (91% vs. 57%, p<0.001), especially in chronic infections (88% vs. 48%, p<0.001.) In acute infections, both tests detected all infection cases. Processing turnaround time was shorter in Alpha defensin lateral flow test than automated leukocyte count (10 min vs. 2–4 hours). Conclusions. Semi-quantitative alpha defensin test was rapid and highly specific for diagnosing PJI (> 95%). However, sensitivity was limited, especially when applying definition criteria including also low grade infections (SOSSID criteria). Therefore, the alpha defensin lateral flow test does not allow a reliable exclusion of PJI, especially not in chronic infections but may be used as confirmatory test

Aim. Diagnosing or excluding a chronic prosthetic joint infection (PJI) prior to revision surgery can be a clinical challenge. To enhance accuracy of diagnosis, several biomarkers were introduced in recent years, but most are either expensive or not available as a rapid test. We compared the diagnostic accuracy of leucocyte esterase (€0.20 per sample), calprotectin (€20 per sample) and alpha defensin (€200 per sample). Method. We prospectively evaluated PJI patients with chronic pain with or without prosthetic loosening between 2017 and 2018. Synovial fluid was collected prior to revision surgery. Leucocyte esterase was measured using a reagent strip (2+ considered as positive), and calprotectin and alpha defensin were measured using a lateral flow immunoassay. Intraoperative cultures (5 periprosthetic tissue samples, synovial fluid and sonication fluid) incubated for 9 days, were used as gold standard. At least two positive cultures of low-grade microorganisms with the same antibiogram were required to diagnose PJI. Results. A total of 19 patients were included (knee =11, hip =8). None of the patients were treated with antibiotics prior to revision surgery. A PJI was diagnosed in 8 patients (42.1%). The diagnostic accuracy of leucocyte esterase vs. calprotectin vs. alpha defensin was as follows; sensitivity 50.0% vs. 87.5% vs. 87.5%, specificity 81.8% vs. 90.9% vs. 100%, positive predictive value 60.0% vs. 87.5% vs. 100% and negative predictive value 75.0% vs. 90.9% vs. 91.6%, respectively. Both calprotectin and alpha defensin were false negative in one PJI caused by Cutibacterium acnes. The other two C. acnes PJIs were correctly diagnosed with both tests. Conclusions. Calprotectin is as accurate as alpha defensin in excluding a chronic PJI at 10% of the costs. Future studies with a large number of patients are necessary to analyze its diagnostic accuracy in very low-grade infections, in particularly caused by C. acnes