The diagnosis of periprosthetic joint infection may be difficult with patients presenting months or years after initial surgery with surgery-associated or haematogenously spread bacteria. Synovasure™ is a new point of care assay that measures alpha defensin produced by activated leucocytes in joints; it is licensed for the diagnosis of periprosthetic joint infections. We sought to include alpha defensin testing in a testing algorithm to improve the diagnosis of periprosthetic joint infection. An algorithm for testing patients with suspected periprosthetic joint infection was developed and agreed among knee surgeons in Gloucestershire, UK. Data was prospectively collected on all tests performed along with information on how the results of the alpha defensin test altered patient management. A sample of joint fluid was taken using aseptic technique in theatre and tested for alpha defensin production at the point of care. Samples were then referred for standard culture and selected samples for 16SrRNA PCR. 12 patients were assayed for alpha defensin in periprosthetic joint fluid during 2015. 7 patients were female, 5 male and ages ranged 64–86 years. 10 patients had a negative point of care alpha defensin test. Only 2 of these patients also had a leukeocyte esterase (LE) test performed and these were negative. The culture results from all samples were negative for both direct and enrichment cultures. 3 samples also had 16SrRNA PCR performed and these were negative. 2 patient samples tested positive for alpha defensin. LE tests were not performed. Both samples were culture negative on direct and enrichment culture however both samples were also referred for 16SrRNA PCR which detected DNA compatible with Staphylococcus caprae/capitis/ saccharolyticus/epidermidis from 1 patient and DNA with homology to Streptococcus gallolyticus/equinusI for the other. Alpha defensin testing improved the diagnosis of prosthetic joint infection. A positive alpha defensin test may be used to select patients for whom 16SrRNA PCR testing is useful in order to maximise the potential for pre-revision infection diagnosis and the planning of appropriate adjuncts such as antibiotic laden cement or calcium sulphate beads. Negative alpha defensin tests on aspirated joint fluid may avoid the need for arthroscopy and biopsy and allow planning for single stage revision surgery without concern for infection

Aim. Diagnosing periprosthetic joint infection after total joint arthroplasty is often challenging. The alpha defensin test has been recently reported as a promising diagnostic test for periprosthetic joint infection. The goal of this study was to determine the diagnostic accuracy of alpha defensin testing. Method. One hundred and eighty-three synovial alpha defensin and synovial fluid C-reactive protein (CRP) tests performed in 183 patients undergoing evaluation for periprosthetic joint infection were reviewed. Results were compared with the Musculoskeletal Infection Society (MSIS) criteria for periprosthetic joint infection. Results. Alpha defensin tests were performed prior to surgical treatment for infection, and 37 of these patients who had these tests were diagnosed by MSIS criteria as having infections. Among this group, the alpha defensin test had a sensitivity of 81.1% (95% confidence interval [CI], 64.8% to 92.0%) and a specificity of 95.9% (95% CI, 91.3% to 98.5%). There were 6 false-positive results, 4 of which were associated with metallosis. There were 7 false negatives, all of which were associated with either draining sinuses (n = 3) or low-virulence organisms (n = 4). A combined analysis of alpha defensin and synovial fluid CRP tests was performed in which a positive result was represented by a positive alpha defensin test and a positive synovial fluid CRP test (n = 28). Among this group, the sensitivity was calculated to be 73.0% (95% CI, 55.9% to 86.2%) and the specificity was calculated to be 99.3% (95% CI, 96.2% to 99.9%). An additional combined analysis was performed where a positive result was represented by a positive alpha defensin test or positive synovial fluid CRP test (n = 64). Among this group, the sensitivity was calculated tobe91.9%(95%CI, 78.1%to98.3%) andthe specificitywas calculated tobe79.5%(95%CI, 72.0%to85.7%). Conclusions. Alpha defensin in combination with synovial fluid CRP demonstrates very high sensitivity for diagnosing periprosthetic joint infection, but may yield false-positive results in the presence of metallosis or false-negative results in the presence of low-virulence organisms. When both alpha defensin and synovial fluid CRP tests are positive, there is a very high specificity for diagnosing periprosthetic joint infection

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

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. To evaluate a panel of peripheral blood and synovial fluid biomarkers for the identification of periprosthetic joint infection PJI. Method. Peripheral blood and synovial fluid measurements of CD64, IL-1a, IL-1b, IL-6, IL-8, IL-10, IL-17, Alpha Defensin and CRP were made on samples collected from patients with suspected PJI using a combination of flow cytometry (CD64), ELISA (Alpha Defensin) and MSD Electrochemiluminescence (IL-1a, IL-1b, IL-6, IL-8, IL-10, IL-17). Receiver operating characteristic (ROC) curves which combine sensitivity and specificity were created for each marker using GraphPad PRISM statistical software. The diagnosis of infection was based on MSIS major criteria. Results. A total of 35 infections were identified (12 acute, 23 chronic). The best performing peripheral blood biomarker in both acute and chronic PJI was CRP with an area under the curve (AUC) of 0.88 (sensitivity 83%, specificity 94%) in acute infection and 0.82 in chronic infection (sensitivity 80%, specificity 85%). In synovial fluid the best performing acute infection marker was CRP with an AUC of 0.94 (sensitivity 87.5%, specificity 95%) and in chronic cases was Alpha defensin with an AUC of 0.98 (sensitivity 100%, specificity 85%). Conclusions. CRP measured in peripheral blood shows excellent diagnostic characteristics in both acute and chronic cases. This is also replicated in synovial fluid from acute PJIs but not in chronic infection where Alpha defensin showed the best performance

Purpose. Unexpected-positive-intraoperative-cultures (UPIC) in presumed aseptic revision-total-knee-arthroplasties (rTKA) are common, and the clinical significance is not entirely clear. In contrast, in some presumably septic rTKA, an identification of an underlying pathogen was not possible, so called unexpected-negative-intraoperative-cultures (UNIC). The purpose of this study was to evaluate alpha defensin (AD) levels in these patient populations. Methods. In this retrospective analysis of our prospectively maintained biobank, we evaluated synovial AD levels from 143 rTKAs. The 2018-Musculoskeletal Infection Society score (MSIS) was used to define our study groups. Overall, 20 rTKA with UPIC with a minimum of one positive intraoperative culture with MSIS 2-≥6 and 14 UNIC samples with MSIS≥6 were compared to 34 septic culture-positive samples (MSIS ≥6) and 75 aseptic culture-negative (MSIS 0–1) rTKAs. Moreover, we compared the performance of both AD-lateral-flow-assay (ADLF) and an enzyme-linked-immunosorbent-assay (ELISA) to test the presence of AD in native and centrifuged synovial fluid. Concentration of AD determined by ELISA and ADLF methods, as well as microbiological, and histopathological results, serum and synovial parameters along with demographic factors were considered. Results. AD was detected in 31/34 (91.2%) samples from the infected-group and in 14/14 (100%) samples in the UNIC group. All UPIC samples showed a negative AD result. Positive AD samples were highly (p<0.001) associated with culture positive and infection related histopathological results. Moreover, we found significantly (p=0.001) more high-virulent microorganisms 19/34 (55.9%) in the infected-group compared to the UPIC-group (0/20). Samples from the infected group with high virulent microorganisms 17/19 (89.5%) showed a positive AD. The presence of methicillin resistant Staphylococcus epidermis (MRSE) led to increased AD (p=0.003) levels when compared to those determined in samples positive for methicillin susceptible S. epidermdis (MSSE). ELISA and ADLF tests were positive with centrifuged (8/8) and native (8/8) synovial fluid. Conclusion. AD showed a solid diagnostic performance in infected and non-infected revisions, and it provided an additional value in the diagnostic of UPIC and UNIC associated to rTKAs. AD levels produced by patients with PJIs caused by high-virulent microorganisms and MRSE are significantly higher compared to those in patients with PJIs caused by either low-virulent or antibiotic susceptible microorganisms. Centrifugation of synovial fluid had no influence in the outcome of ADLF quantification. Keywords: Alpha-defensin, UPIC, UNIC, revision-knee-arthroplasty

Aim. Apart from other biomarkers isolated in the synovial fluid, alpha-defensin appears to be a promising diagnostic tool to confirm a periprosthetic joint infection (PJI) in the hip or knee. The purpose of this study was to evaluate the sensitivity and specificity of an alpha defensin lateral flow (ADLF) test compared to usual standard classifications in the diagnostic management of PJI. Method. This investigation was set up as a multicenter prospective cohort study. Synovial fluid was obtained by means of joint aspiration or intra-operative tissue biopsies. A presumptive PJI diagnosis was made according to criteria outlined by the Musculoskeletal Infection Society (MSIS), the Infectious Diseases Society of America (IDSA) and the European Bone and Joint Infection Society (EBJIS). The intention to treat by the surgeon was logged. Sensibility and specificity for the ADLF test was plotted for each aforementioned diagnostic algorithm. Spearman correlations between all scores were analyzed. Multiple logistic regression was used to determine the contribution of independent variables to the probability of PJI. Results. Hundred thirty-six patients with a painful arthroplasty were assessed for infection and rated by the treating surgeon as potentially infected or not on the basis of clinical and laboratory information. According to the EBJIS criteria sixty-eight patients were deemed infected, fifty according to the IDSA criteria, forty-one according to the MSIS criteria and forty according to the ADLF test. However, the sensitivity of ADLF test was 87.8% for MSIS, 70% for IDSA and 55.8% for EBJIS. The specificity of ADLF test was between 94% – 97%. Good correlation was observed between synovial fluid culture and ADLF test (r = 0.73). Low to excellent correlations between the ADLF test and the EBJIS (r = 0.58), IDSA (r = 0.68), and MSIS score (r = 0.84) were observed. The surgeon's intention to treat correlated well with the MSIS score (r = 0.86), and moderately with the EBJIS (r = 0.59). Conclusions. ADLF test sensibility was variable, but its specificity was excellent. Most of the cases, not retained by MSIS but classified by EBJIS as infected, got a negative microbiological result. Considering an accepted 20% negative microbiological result rate in PJI diagnostic, EBJIS is clearly overestimating the number of infected cases. MSIS score correlates with the surgeon intention to treat and ADLF test

The AAOS clinical practice guideline for diagnosis of periprosthetic joint infection (PJI) and the MSIS definition of PJI were both “game changers” in terms of diagnosing PJI and the reporting of outcomes for research. However, the introduction of new diagnostic modalities, including biomarkers, prompted a re-look at the diagnostic criteria for PJI. Further there was a desire to develop an evidence-based, validated algorithm for the diagnosis of PJI. This multi-institutional study led by Dr. Jay Parvizi examined revision total joint arthroplasty patients from three academic institutions. For development of the algorithm, infected and aseptic cohorts were defined. PJI cases were defined using only the major criteria from the Musculoskeletal Infection Society (MSIS) definition (n=684). Aseptic cases underwent revision for a non-infective indication and did not show evidence of PJI or undergo a reoperation for any reason within 2 years (n=820). Risk factors, clinical findings, serum and synovial markers as well as intraoperative findings were assessed. A stepwise approach using random forest analysis and multivariate regression was used to generate relative weights for each of the various variables assessed at each stage to create an algorithm for diagnosing PJI using the 3 most important tests from each step. The algorithm was formally validated on a separate cohort of 422 patients, 222 who were treated with a 2-stage exchange for PJI who subsequently failed secondary to PJI within one year and 200 patients who underwent revision surgery for an aseptic diagnosis and had no evidence of PJI within two years and did not undergo a reoperation for any reason. The first step in evaluating PJI should include a physical examination to identify a sinus tract, followed by serum testing for C-reactive protein (cut-off value 1mg/dl), D-dimer (cut-off value 860ng/mL) and/or erythrocyte sedimentation rate (cut-off value 30mm/hr) in that order of importance. If at least one of these are elevated, or if there is a high clinical suspicion, joint aspiration should be performed, sending the fluid obtained for a synovial fluid white blood-cell (cut-off value 3,000 wbc/uL) or leukocyte esterase strip testing, polymorphonuclear percentage (cut-off value 80%) and culture. Alpha defensin did not show added benefit as a routine diagnostic test. Major diagnostic criteria are the same whereby the presence of a sinus tract or (2) positive cultures showing the same organism defines PJI. Special care should be taken in cases of ALTR (failed metal-on-metal bearing), crystalline deposition disease, inflammatory arthritis flares or slow growing organisms. In the rare cases where no fluid is obtained at the time of an attempted aspiration and revision surgery is not planned, then this is the rare scenario where nuclear imaging (my preference is an indium labeled white blood cell scan) or a biopsy can be performed. The updated definition of PJI demonstrated a higher sensitivity of 97.7% when compared to the MSIS criteria (79.3%) and the ICM definition (86.9%), with a similar specificity of 99.5%. However, just over 2% of patients examined do fall into the “inconclusive” category. The proposed diagnostic algorithm demonstrated a high overall sensitivity (96.9%) and specificity (99.5%)

Periprosthetic joint infection (PPJI) following shoulder arthroplasty is uncommon, with an overall rate of 0.98%. However, the rates following revision arthroplasty and reverse arthroplasty are much higher. Given the rapid increase in the prevalence of shoulder arthroplasty and the increasing revision burden, the cost of PPJI to society will likely increase substantially. The most common organisms found in PPJI following shoulder arthroplasty are Staphylococcus aureus, coagulase-negative Staphylococcus, and Propionibacterium acnes (P. acnes). P. acnes is especially common in males. Traditional testing for PPJI includes aspiration, white blood cell count (WBC), erythrocyte sedimentation rate (ESR), and c-reactive protein (CRP). Aspiration often yields a dry tap and when fluid is obtained for culture, a positive result is helpful but a negative result does not rule out PPJI. Although WBC, ESR, and CRP are often positive with PPJI in the lower extremity, they are most often negative in shoulder PPJI. Although bone scans and WBC labeled scans are used, they are expensive and have low sensitivity and specificity. New testing and techniques have been reported in an attempt to improve sensitivity and specificity for PPJI. These techniques can be divided into tests on serum, synovial fluid, and tissue. Serum Interleukin-6 (IL-6) is highly specific (94%) for shoulder PPJI but has low sensitivity (14%). Synovial fluid can be tested for leukocyte esterase using a simple and cheap technique. In lower extremity PPJI it has shown to be helpful. It is not as helpful in shoulder PPJI with 30% sensitivity and 67% specificity. Alpha defensin has been reported to be more sensitive (63%) and as specific (95%) as traditional techniques but still lacks predictive value. Testing for specific cytokines (IL-2, IL-6, TNF- α) within synovial fluid is not widely used as yet but has shown promise with 80% sensitivity and 90% specificity. Obtaining tissue for culture and other testing is probably the most reliable way of confirming PPJI for the shoulder. Frozen sections taken at the time of revision can be helpful but is very pathologist dependent and institution specific. With a dedicated musculoskeletal pathologist, the finding of 10 or more WBCs per high powered field has been reported to be 72% sensitive and 100% specific for P. acnes and 63% sensitive and 100% specific for other organisms. Cultures from arthroscopic tissue biopsy have also been found to have high sensitivity (100%) and specificity (100%). Genetic testing of tissue biopsy specimens (PCR/NGS) has recently been reported and shows great promise. The significance of positive cultures and other tests, especially for P. acnes is unclear. There is a high rate of positive intra-operative cultures in primary cases of shoulder arthroplasty. In addition, intra-operative cultures taken at the time of revision, even in cases in which infection is not suspected, are frequently positive for P. acnes with weak correlation with rates of post-operative clinical infection. In conclusion, shoulder PPJI is a difficult problem to deal with. The definition of shoulder PPJI is currently unclear and further study is needed. There is no ideal test to confirm it. A reasonable approach is to aspirate for culture, and perform serum tests for WBC, ESR, and C-reactive protein. If any of these is positive in the setting of a painful arthroplasty, PPJI should be assumed until proven otherwise. Operative tissue cultures are probably the most reliable test but the clinical significance is not always obvious. Synovial fluid cytokine profiles and tissue PCR/NGS show promise for the future

Introduction. In revision surgery, detection of periprosthetic joint infection is of prime importance. Valuable preoperative and intraoperative diagnostic tests and tools are necessary. The classical standard procedures are puncture and bacteriology examination, frozen section intraoperative and powerfield micro analysis. Since autumn 2014 a new device for detection of periprosthetic joint infection is available, named Synovasure. It is a fast test for the detection of Alpha defensing, which plays a major role in the antimicrobial defence and only occurs in inflammatory processes. „The alpha-defensin test is an immunoassay that measures the concentration of the alpha-defensin peptide in human synovialfluid. A-Defensin is an antimicrobial peptide that is secreted into the synovial fluid by human cells in response to pathogenic presence” (Deirmengian C et al., CORR 2014). Summarized, the evidence of Alpha defensin indicates infection. It is produced by CD Diagnostics (Wynnewood, PA, USA) and merchandised by Zimmer (Warsaw, IL, USA). We are using Synovasure in daily routine at our department since September 2014. The aim of this conducted study is to present our first clinical experience and to report our results of the first 54 cases. Material and Methods. At our department Synovasure is standardly used in hip and knee revision surgery. Additionally an intraoperative frozen section and a standard bacteriology were performed. The explanted endprosthesis were sent to examination by sonification in order to gain culture of the sonification fluid and were further examined by Multiplex PCR. A pathologist with more than 15 years of experience conducted the frozen section. The results of Synovasure were matched with all above examinations in order to describe specifity and sensitivity of it. Results. A negative Synovasure Test during surgery and a negative PCR were observed in 3 patients, however, the bacterial culture was positive (after 14 days of breeding) as well as the Multiplex PCR. One patient had a negative frozen section and a negative culture but a positive PCR. Another patient with a high CRP level, all clinical signs of infection and a positive Synovasure Test, had 6 negative cultures. This patient suffered from a Metallosis as well, due to a broken PE inlay of the TKA, which supports the previously stated that Metallosis may interfere this new tool. Unfortunately in this patient neither a frozen section nor a PCR are available. One patient, who had explanation due to infection, underwent reimplantation. During surgery the Synovasure Test and the frozen section were negative (Synovial Fluid), but postoperatively a positive culture and a positive histological report for infection were assessed. Furthermore, a total of 5 tests showed an application error and the test did not show any control line. Conclusion. In conclusion Synovasure helps to detect perprosthetic joint infection in an easy and fast manner. It is simple to integrate into daily routine, nevertheless all standardized examinations for infection need to be conducted