The False-Positive Rate of Synovial Fluid Culture at a Single Clinical Laboratory Using Culture Bottles.

Introduction Synovial fluid (SF) cultures can yield false-positive or negative results when diagnosing periprosthetic joint infection (PJI). False-positives may arise during sample collection or from laboratory contamination. Understanding false-positive SF culture rates is crucial for interpreting PJI laboratory data, yet clinical laboratories rarely report these rates. This study aimed to define the false-positive SF culture rate at a major specialized clinical laboratory. Methods This study retrospectively analyzed prospectively collected data at a single clinical laboratory that receives SF for clinical testing for PJI. A total of 180,317 periprosthetic SF samples from the hip, knee, and shoulder were identified from January 2016 to December 2023, which met the inclusion criteria for this study. Samples were classified by both a modified 2018 International Consensus Meeting (ICM) score and an inflammation score that combined the SF-C-reactive protein, alpha-defensin, SF-white blood cell count, and SF-polymorphonuclear% into one standardized metric. Logistic regression was utilized to evaluate the impact of various collection-based characteristics on culture positivity, including inflammation biomarkers, the source joint, quality control metrics, and days of specimen transport to the laboratory. SF culture false-positivity was calculated based on the ICM category of "not-infected" or low inflammation score. Results Overall, 13.3% (23,974/180,317) of the samples were associated with a positive culture result: 12.5% for knee samples, 20.3% for hip samples, and 14.7% for shoulder samples. The false-positive SF culture rate among 131,949 samples classified as "not-infected" by the modified 2018 ICM definition was 0.47% (95%CI: 0.43 to 0.51%). Stratification by joint revealed a false-positive rate of 0.34% (95%CI: 0.31 to 0.38%) for knee samples, 1.24% (95%CI: 1.05 to 1.45%) for hip samples, and 3.02% (95%CI: 2.40 to 3.80%) for shoulder samples, with p < 0.0001 for all comparisons. The false-positive SF culture rate among 90,156 samples, representing half of all samples with the lowest standardized inflammation scores, was 0.47% (95%CI: 0.43 to 0.52%). Stratification by joint revealed a false-positive rate of 0.33% (95%CI: 0.29 to 0.37%) for knee samples, 1.45% (95%CI: 1.19 to 1.77%) for hip samples, and 3.09% (95%CI: 2.41 to 3.95%) for shoulder samples, with p<0.0001 for all comparisons. Multivariate logistic regression demonstrated the joint source (hip, shoulder) and poor sample quality as collection-based factors associated with a false-positive culture. Evaluation of a cohort of samples selected to minimize collection-based causes of false-positive culture demonstrated a false-positive rate of 0.30%, representing the ceiling limit for laboratory-based SF culture false-positivity. Conclusions This study utilizes two methods to estimate the false-positive SF culture rate at a single specialized clinical laboratory, demonstrating an overall false-positive rate of approximately 0.5%. Stratification of samples by source joint demonstrated that periprosthetic SF from the shoulder and hip have a substantially higher false-positive culture rate than that of the knee. The lowest false-positive SF culture rate (0.30%) was observed among samples from the knee-passing quality control. Culture positivity due to contamination at this specific laboratory is less than 0.30% because all specimens undergo identical processing.

Synovial Fluid C-reactive Protein Clinical Decision Limit and Diagnostic Accuracy for Periprosthetic Joint Infection.

Introduction C-reactive protein (CRP) has long served as a prototypical biomarker for periprosthetic joint infection (PJI). Recently, synovial fluid (SF)-CRP has garnered interest as a diagnostic tool, with several studies demonstrating its diagnostic superiority over serum CRP for the diagnosis of PJI. Although previous studies have identified diagnostic thresholds for SF-CRP, they have been limited in scope and employed various CRP assays without formal validation for PJI diagnosis. This study aimed to conduct a formal single clinical laboratory validation to determine the optimal clinical decision limit of SF-CRP for the diagnosis of PJI. Methods A retrospective analysis of prospectively collected data was performed using receiver operating characteristic (ROC) and area under the curve (AUC) analyses. Synovial fluid samples from hip and knee arthroplasties, received from over 2,600 institutions, underwent clinical testing for PJI at a single clinical laboratory (CD Laboratories, Zimmer Biomet, Towson, MD) between 2017 and 2022. Samples were assayed for SF-CRP, alpha-defensin, white blood cell count, neutrophil percentage, and microbiological culture. After applying selection criteria, the samples were classified with the 2018 ICM PJI scoring system as "infected," "not infected," or "inconclusive." Data were divided into training and validation sets. The Youden Index was employed to optimize the clinical decision limit. Results A total of 96,061 samples formed the training (n = 67,242) and validation (n = 28,819) datasets. Analysis of the biomarker median values, culture positivity, anatomic distribution, and days from aspiration to testing revealed nearly identical specimen characteristics in both the training set and validation set. SF-CRP demonstrated an AUC of 0.929 (95% confidence interval (CI): 0.926-0.932) in the training set, with an optimal SF-CRP clinical decision limit for PJI diagnosis of 4.45 mg/L. Applying this cutoff to the validation dataset yielded a sensitivity of 86.1% (95% CI: 85.0-87.1%) and specificity of 87.1% (95% CI: 86.7-87.5%). No statistically significant difference in diagnostic performance was observed between the validation and training sets. Conclusion This study represents the largest single clinical laboratory evaluation of an SF-CRP assay for PJI diagnosis. The optimal CRP cutoff (4.45 mg/L) for PJI, which yielded a sensitivity of 86.1% and a specificity of 87.1%, is specific to the assay methodology and laboratory performing the assay. We propose that an SF-CRP test with a laboratory-validated optimal clinical decision limit for PJI may be preferable, in a clinical diagnostic setting, to serum CRP tests that do not have laboratory-validated clinical decision limits for PJI.

Achieving High Accuracy in Predicting the Probability of Periprosthetic Joint Infection From Synovial Fluid in Patients Undergoing Hip or Knee Arthroplasty: The Development and Validation of a Multivariable Machine Learning Algorithm.

Background and objective The current periprosthetic joint infection (PJI) diagnostic guidelines require clinicians to interpret and integrate multiple criteria into a complex scoring system. Also, PJI classifications are often inconclusive, failing to provide a clinical diagnosis. Machine learning (ML) models could be leveraged to reduce reliance on these complex systems and thereby reduce diagnostic uncertainty. This study aimed to develop an ML algorithm using synovial fluid (SF) test results to establish a PJI probability score. Methods We used a large clinical laboratory's dataset of SF samples, aspirated from patients with hip or knee arthroplasty as part of a PJI evaluation. Patient age and SF biomarkers [white blood cell count, neutrophil percentage (%PMN), red blood cell count, absorbance at 280 nm wavelength, C-reactive protein (CRP), alpha-defensin (AD), neutrophil elastase, and microbial antigen (MID) tests] were used for model development. Data preprocessing, principal component analysis, and unsupervised clustering (K-means) revealed four clusters of samples that naturally aggregated based on biomarker results. Analysis of the characteristics of each of these four clusters revealed three clusters (n=13,133) with samples having biomarker results typical of a PJI-negative classification and one cluster (n=4,032) with samples having biomarker results typical of a PJI-positive classification. A decision tree model, trained and tested independently of external diagnostic rules, was then developed to match the classification determined by the unsupervised clustering. The performance of the model was assessed versus a modified 2018 International Consensus Meeting (ICM) criteria, in both the test cohort and an independent unlabeled validation set of 5,601 samples. The SHAP (SHapley Additive exPlanations) method was used to explore feature importance. Results The ML model showed an area under the curve of 0.993, with a sensitivity of 98.8%, specificity of 97.3%, positive predictive value (PPV) of 92.9%, and negative predictive value (NPV) of 99.8% in predicting the modified 2018 ICM diagnosis among test set samples. The model maintained its diagnostic accuracy in the validation cohort, yielding 99.1% sensitivity, 97.1% specificity, 91.9% PPV, and 99.9% NPV. The model's inconclusive rate (diagnostic probability between 20-80%) in the validation cohort was only 1.3%, lower than that observed with the modified 2018 ICM PJI classification (7.4%; p<0.001). The SHAP analysis found that AD was the most important feature in the model, exhibiting dominance among >95% of "infected" and "not infected" diagnoses. Other important features were the sum of the MID test panel, %PMN, and SF-CRP. Conclusions Although defined methods and tools for diagnosis of PJI using multiple biomarker criteria are available, they are not consistently applied or widely implemented. There is a need for algorithmic interpretation of these biomarkers to enable consistent interpretation of the results to drive treatment decisions. The new model, using clinical parameters measured from a patient's SF sample, renders a preoperative probability score for PJI which performs well compared to a modified 2018 ICM definition. Taken together with other clinical signs, this model has the potential to increase the accuracy of clinical evaluations and reduce the rate of inconclusive classification, thereby enabling more appropriate and expedited downstream treatment decisions.

Mitigating Concerns Over Transport Delays: An Analysis of Synovial Fluid Culture Results in Arthroplasty.

INTRODUCTION:  There is a concern in the field of arthroplasty that synovial fluid transport delays may reduce the accuracy of synovial fluid culture. However, synovial fluid samples collected in the office, and sometimes in a hospital setting, often require transport to a third-party central or specialty laboratory, causing delays in the initiation of culture incubation. This study investigated the impact of transportation delays on synovial fluid culture results. METHODS:  A retrospective review of prospectively collected data at one clinical laboratory, from 2016 to 2022, was conducted. A total of 125,270 synovial fluid samples from knee arthroplasties, from 2,858 different US institutions, were transported to a single clinical laboratory for diagnostic testing including synovial fluid culture (blood culture bottles). Synovial fluid to be cultured was transported in red top tubes without additives. Samples were grouped into six-time cohorts based on the number of days between aspiration and culture initiation (1-day-delay to 6-day-delay). Metrics such as culture positivity, false-positive culture rate, culture sensitivity, and proportional growth of top genera of organisms were assessed across the cohorts. RESULTS:  Of the 125,270 samples in this study, 71.2% were received the day after aspiration (1-day-delay), with an exponential decrease in samples received on each subsequent day. Culture-positive rates for synovial fluid samples received after 1, 2, 3, 4, 5, and 6 days of transport time were 12.2%, 13.3%, 13.5%, 13.1%, 11.6%, and 11.0%, respectively. The maximum absolute difference in culture-positive rate compared to the 1-day-delay cohort was an increase of 1.3% in the 3-day-delay cohort, which was not considered a clinically meaningful difference. The estimated false-positive culture rate remained relatively consistent across time cohorts, with values of 0.3%, 0.4%, 0.3%, 0.2%, 0.5%, and 0.5% for 1, 2, 3, 4, 5, and 6 days of transport time, respectively. None of the cohorts showed a statistically significant difference after adjustment for multiplicity compared to the 1-day-delay cohort. Culture sensitivity was estimated at 68.2%, 67.2%, 70.5%, 70.7%, 65.9%, and 70.7% for 1, 2, 3, 4, 5, and 6 days of transport time, respectively. None of the cohorts showed a statistically significant difference after adjustment for multiplicity compared to the 1-day-delay cohort. Organism proportions were consistent across time cohorts, with Staphylococcus being the most commonly identified organism. No statistically significant differences were found in the proportional contribution of major genera across the cohorts. CONCLUSIONS:  Synovial fluid culture exhibited surprisingly consistent results despite variable transport time to the destination laboratory, with differences that have minimal clinical importance. While the authors of this study advocate for short transport times as a best practice to expedite diagnosis, it appears that concerns regarding the rapid degradation of culture results due to synovial fluid transportation is unwarranted.

Nationwide Results of Microorganism Antigen Testing as a Component of Preoperative Synovial Fluid Analysis.

BACKGROUND: Antigen immunoassays to detect synovial fluid (SF) microorganisms have recently been made available for clinical use. The purpose of this study was to determine the sensitivity and specificity of an SF microorganism antigen immunoassay detection (MID) panel, evaluate the panel's capability to detect microorganisms in the setting of culture-negative periprosthetic joint infection (PJI), and determine diagnostic predictive values of the MID panel for PJI. METHODS: This study included 67,441 SF samples obtained from a hip or knee arthroplasty, from 2,365 institutions across the United States, submitted to 1 laboratory for diagnostic testing. All data were prospectively compiled and then were analyzed retrospectively. Preoperative SF data were used to classify each specimen by the International Consensus Meeting (2018 ICM) definition of PJI: 49,991 were not infected, 5,071 were inconclusive, and 12,379 were infected. The MID panel, including immunoassay tests to detect Staphylococcus, Candida, and Enterococcus, was evaluated to determine its diagnostic performance. RESULTS: The MID panel demonstrated a sensitivity of 94.2% for infected samples that yielded positive cultures for target microorganisms (Staphylococcus, Candida, or Enterococcus). Among infected samples yielding positive cultures for their respective microorganism, individual immunoassay test sensitivity was 93.0% for Staphylococcus, 92.3% for Candida, and 97.2% for Enterococcus. The specificity of the MID panel for samples that were not infected was 98.4%, yielding a false-positive rate of 1.6%. The MID panel detected microorganisms among 49.3% of SF culture-negative infected samples. For PJI as a diagnosis, the positive predictive value of the MID panel was 91.7% and the negative predictive value was 93.8%. Among MID-positive PJIs, 16.2% yielded a discordant cultured organism instead of that detected by the antigen test. CONCLUSIONS: SF microorganism antigen testing provides a timely adjunct method to detect microorganisms in the preoperative SF aspirate, yielding a low false-positive rate and enabling the detection of a microorganism in nearly one-half of SF culture-negative PJIs. LEVEL OF EVIDENCE: Prognostic Level II . See Instructions for Authors for a complete description of levels of evidence.

Extended Release of Bupivacaine from Temperature-responsive Hydrogels Provides Multi-day Analgesia for Postoperative Pain.

OBJECTIVE: A local anesthetic that provides analgesia lasting at least three days could significantly improve postoperative pain management. This study evaluated the analgesic efficacy and safety of an extended-release formulation of bupivacaine based on the injectable hydrogel carrier poly(N-isopropylacrylamide-co-dimethylbutyrolactone acrylamide-co-Jeffamine M-1000 acrylamide) (PNDJ). METHODS: The efficacy of PNDJ containing 4% bupivacaine (SBG004) given by peri-incisional subcutaneous injection (SBG004 SC) or wound filling instillation (SBG004 WF) was evaluated compared to saline, liposomal bupivacaine, bupivacaine collagen sponge, bupivacaine-meloxicam polyorthoester, and bupivacaine HCl in a porcine skin and muscle incision model. Mechanical allodynia was assessed by withdrawal from application of von Frey filaments, and local tolerance was evaluated by histology. Bupivacaine pharmacokinetics for SBG004 SC were measured in rabbits (16.5 mg bupivacaine/kg). RESULTS: Animals demonstrated less mechanical allodynia at incisions receiving SBG004 SC for up to 96 hours postoperatively. Incisions treated with SBG004 SC tolerated more force without a withdrawal indicative of pain compared to saline for 96 hours, and compared to SBG004 WF and all active controls at 24, 48, and 72 hours except bupivacaine-meloxicam polyorthoester at 72 hours. By 49 days, SBG004 was histologically absent and was replaced with granulation tissue infiltrated with immune cells in some areas. In rabbits, Cmax was 41.6 ± 9.7 ng/mL with t1/2 82.0 ± 35.8 hours (mean ± SD). CONCLUSIONS: Peri-incisional SBG004 SC provided extended release of bupivacaine sufficient to reduce sensation of incisional pain for 96 hours, in vivo bupivacaine delivery for at least 7 days, and a favorable local and systemic toxicity profile.

Physician Use of Multiple Criteria to Diagnose Periprosthetic Joint Infection May Be Less Accurate Than the Use of an Individual Test.

Introduction Multiple-criterion scoring systems for periprosthetic joint infection (PJI) can be algorithmically implemented in research, diagnostically outperforming individual tests. This improved performance may be lost in the practice setting, where clinicians rarely utilize strict algorithms. The ability of physicians to interpret multiple criteria for PJI and confront the complexity of combining them into a final diagnosis has never been studied. This study assessed the diagnostic characteristics of physicians using multiple criteria to diagnose PJI and compared the physicians' diagnostic accuracy to that of individual tests. Methods A total of 12 physicians, including academic arthroplasty surgeons (N=4), community arthroplasty surgeons (N=4), and infectious disease (ID) specialists (N=4) were asked to use their routine clinical diagnostic practice to assign a diagnosis to 277 clinical vignettes using multiple preoperative laboratory criteria for PJI. The undecided rate, interobserver agreement, and accuracy of physicians were characterized relative to the 2013 Musculoskeletal Infection Society (MSIS) gold standard and compared to the accuracy of each individual laboratory test for PJI. Results Physicians interpreting multiple criteria for PJI demonstrated high undecided diagnosis rates (mean=23.5%), poor interobserver agreement (kappa range=0.49-0.63), and mean accuracy of 90.8% (range:85.8%-97.4%) compared to the 2013 MSIS gold standard. The group of academic arthroplasty surgeons had a lower rate of undecided diagnoses than community arthroplasty surgeons (16.2% vs. 29.1%; p<0.0001) or ID specialists (16.2% vs. 25.1%; p<0.0001). Academic arthroplasty surgeons also exhibited a higher interobserver agreement than community arthroplasty surgeons (kappa = 0.63 (95%CI:0.59-0.68) vs. 0.49 (95%CI:0.44-0.54)). Mean physician accuracy (90.8%) was inferior to the alpha-defensin laboratory test (96.0%;p=0.0034) and the alpha-defensin lateral-flow test (94.6%;p=0.036), comparable to synovial fluid white blood cells (SF-WBC) (93.3%;p=0.17) and synovial fluid polymorphonuclear cell % (SF-PMN%) (94.0%;p=0.11), and superior to the erythrocyte sedimentation rate (ESR) (86.2%;p<0.0001) and C-reactive protein (CRP) (84.6%;p<0.0001). Only two academic arthroplasty surgeons in this study were able to outperform every individual test for PJI by combining multiple criteria to make a diagnosis. Conclusion Although multiple-criterion scoring systems may outperform individual tests for diagnosing PJI in the research setting, it appears that the complexity of using multiple tests to diagnose PJI causes indecision and variability among physicians. Physician use of multiple preoperative criteria to diagnose PJI is less accurate than the strict algorithmic calculation of the diagnosis as achieved in research. In fact, most physicians in this study would have improved their diagnostic accuracy for PJI by simply utilizing a single good test to make the diagnosis, instead of trying to combine multiple tests into a decision. We propose that less complex diagnostic criteria should be explored for routine clinical utilization.

Local antimicrobial delivery from temperature-responsive hydrogels reduces incidence of intra-abdominal infection in rats.

The objective of this study was to evaluate local antimicrobial delivery from temperature-responsive hydrogels for preventing infection in a rat model of intra-abdominal infection (IAI), and to determine whether delivery of tobramycin and vancomycin in combination is effective against IAI pathogens. Rats received intraperitoneal inoculation of E. coli, rat cecal contents, or cecal contents supplemented with E. coli, and received either no treatment, subcutaneous cefoxitin, or local delivery from hydrogels containing vancomycin, tobramycin, or both antimicrobials. Only the hydrogel with tobramycin and vancomycin significantly increased the infection free-rate compared to no treatment for all inocula (E. coli: 13/17, p < 0.0001; cecal contents: 11/17, p = 0.0013; cecal contents + E. coli: 15/19, p < 0.0001). Additionally, tobramycin and vancomycin displayed no synergy or antagonism against clinical isolates in vitro. Local delivery of tobramycin and vancomycin from temperature-responsive hydrogels provides broad coverage and high antimicrobial concentrations for several hours that may be effective for preventing IAIs.

In vivo evaluation of temperature-responsive antimicrobial-loaded PNIPAAm hydrogels for prevention of surgical site infection.

Surgical site infections (SSIs) are a persistent clinical challenge. Local antimicrobial delivery may reduce the risk of SSI by increasing drug concentrations and distribution in vulnerable surgical sites compared to what is achieved using systemic antimicrobial prophylaxis alone. In this work, we describe a comprehensive in vivo evaluation of the safety and efficacy of poly(N-isopropylacrylamide-co-dimethylbutyrolactone acrylamide-co-Jeffamine M-1000 acrylamide) [PNDJ], an injectable temperature-responsive hydrogel carrier for antimicrobial delivery in surgical sites. Biodistribution data indicate that PNDJ is primarily cleared via the liver and kidneys following drug delivery. Antimicrobial-loaded PNDJ was generally well-tolerated locally and systemically when applied in bone, muscle, articulating joints, and intraperitoneal space, although mild renal toxicity consistent with the released antimicrobials was identified at high doses in rats. Dosing of PNDJ at bone-implant interfaces did not affect normal tissue healing and function of orthopedic implants in a transcortical plug model in rabbits and in canine total hip arthroplasty. Finally, PNDJ was effective at preventing recurrence of implant-associated MSSA and MRSA osteomyelitis in rabbits, showing a trend toward outperforming commercially available antimicrobial-loaded bone cement and systemic antimicrobial administration. These studies indicate that antimicrobial-loaded PNDJ hydrogels are well-tolerated and could reduce incidence of SSI in a variety of surgical procedures.

Adjuvant antibiotic-loaded bone cement: Concerns with current use and research to make it work.

Antibiotic-loaded bone cement (ALBC) is broadly used to treat orthopaedic infections based on the rationale that high-dose local delivery is essential to eradicate biofilm-associated bacteria. However, ALBC formulations are empirically based on drug susceptibility from routine laboratory testing, which is known to have limited clinical relevance for biofilms. There are also dosing concerns with nonstandardized, surgeon-directed, hand-mixed formulations, which have unknown release kinetics. On the basis of our knowledge of in vivo biofilms, pathogen virulence, safety issues with nonstandardized ALBC formulations, and questions about the cost-effectiveness of ALBC, there is a need to evaluate the evidence for this clinical practice. To this end, thought leaders in the field of musculoskeletal infection (MSKI) met on 1 August 2019 to review and debate published and anecdotal information, which highlighted four major concerns about current ALBC use: (a) substantial lack of level 1 evidence to demonstrate efficacy; (b) ALBC formulations become subtherapeutic following early release, which risks induction of antibiotic resistance, and exacerbated infection from microbial colonization of the carrier; (c) the absence of standardized formulation protocols, and Food and Drug Administration-approved high-dose ALBC products to use following resection in MSKI treatment; and (d) absence of a validated assay to determine the minimum biofilm eradication concentration to predict ALBC efficacy against patient specific micro-organisms. Here, we describe these concerns in detail, and propose areas in need of research.

Bacterial toxins in musculoskeletal infections.

Musculoskeletal infections (MSKIs) remain a major health burden in orthopaedics. Bacterial toxins are foundational to pathogenesis in MSKI, but poorly understood by the community of providers that care for patients with MSKI, inducing an international group of microbiologists, infectious diseases specialists, orthopaedic surgeons and biofilm scientists to review the literature in this field to identify key topics and compile the current knowledge on the role of toxins in MSKI, with the goal of illuminating potential impact on biofilm formation and dispersal as well as therapeutic strategies. The group concluded that further research is needed to maximize our understanding of the effect of toxins on MSKIs, including: (i) further research to identify the roles of bacterial toxins in MSKIs, (ii) establish the understanding of the importance of environmental and host factors and in vivo expression of toxins throughout the course of an infection, (iii) establish the principles of drug-ability of antitoxins as antimicrobial agents in MSKIs, (iv) have well-defined metrics of success for antitoxins as antiinfective drugs, (v) design a cocktail of antitoxins against specific pathogens to (a) inhibit biofilm formation and (b) inhibit toxin release. The applicability of antitoxins as potential antimicrobials in the era of rising antibiotic resistance could meet the needs of day-to-day clinicians.

Biophysical differentiation of susceptibility and chemical differences in Staphylococcus aureus.

Differentiating bacteria strains using biophysical forces has been the focus of recent studies using dielectrophoresis (DEP). The refinement of these studies has created high-resolution separations such that very subtle properties of the cells are enough to induce significant differences in measurable biophysical properties. These high-resolution capabilities build upon the advantages of DEP which include small sample sizes and fast analysis times. Studies focusing on differentiating antimicrobial resistant and susceptible bacteria potentially have significant impact on human health and medical care. A prime example is Staphylococcus aureus, which commonly colonizes adults without ill effects. However, the pathogen is an important cause of infections, including surgical site infections. Treatment of S. aureus infections is generally possible with antimicrobials, but antimicrobial resistance has emerged. Of special importance is resistance to methicillin, an antimicrobial created in response to resistance to penicillin. Here, dielectrophoresis is used to study methicillin-resistant (MRSA) and -susceptible S. aureus (MSSA) strains, both with and without the addition of a fluorescent label. The capture onset potential of fluorescently-labeled MRSA (865 ± 71 V) and thus the ratio of electrokinetic to dielectrophoretic mobility, was found to be higher than that of fluorescently-labeled MSSA (685 ± 61 V). This may be attributable to the PBP2a enzyme present in the MRSA strain and not in the MSSA bacteria. Further, unlabeled MRSA was found to have a capture onset potential of 732 ± 44 V, while unlabeled MSSA was found to have a capture onset potential of 562 ± 59 V. This shows that the fluorescently-labeled bacteria require a higher applied potential, and thus ratio of mobilities, to capture than the unlabeled bacteria.

2018 International Consensus Meeting on Musculoskeletal Infection: Research Priorities from the General Assembly Questions.

Musculoskeletal infections (MSKI) remain the bane of orthopedic surgery, and result in grievous illness and inordinate costs that threaten healthcare systems. As prevention, diagnosis, and treatment has remained largely unchanged over the last 50 years, a 2nd International Consensus Meeting on Musculoskeletal Infection (ICM 2018, https://icmphilly.com) was completed. Questions pertaining to all areas of MSKI were extensively researched to prepare recommendations, which were discussed and voted on by the delegates using the Delphi methodology. The questions, including the General Assembly (GA) results, have been published (GA questions). However, as critical outcomes include: (i) incidence and cost data that substantiate the problems, and (ii) establishment of research priorities; an ICM 2018 research workgroup (RW) was assembled to accomplish these tasks. Here, we present the result of the RW consensus on the current and projected incidence of infection, and the costs per patient, for all orthopedic subspecialties, which range from 0.1% to 30%, and $17,000 to $150,000. The RW also identified the most important research questions. The Delphi methodology was utilized to initially derive four objective criteria to define a subset of the 164 GA questions that are high priority for future research. Thirty-eight questions (23% of all GA questions) achieved the requisite > 70% agreement vote, and are highlighted in this Consensus article within six thematic categories: acute versus chronic infection, host immunity, antibiotics, diagnosis, research caveats, and modifiable factors. Finally, the RW emphasizes that without appropriate funding to address these high priority research questions, a 3rd ICM on MSKI to address similar issues at greater cost is inevitable.

Temperature-responsive PNDJ hydrogels provide high and sustained antimicrobial concentrations in surgical sites.

Local antimicrobial delivery is a promising strategy for improving treatment of deep surgical site infections (SSIs) by eradicating bacteria that remain in the wound or around its margins after surgical debridement. Eradication of biofilm bacteria can require sustained exposure to high antimicrobial concentrations (we estimate 100-1000 µg/mL sustained for 24 h) which are far in excess of what can be provided by systemic administration. We have previously reported the development of temperature-responsive hydrogels based on poly(N-isopropylacrylamide-co-dimethylbutyrolactone acrylate-co-Jeffamine M-1000 acrylamide) (PNDJ) that provide sustained antimicrobial release in vitro and are effective in treating a rabbit model of osteomyelitis when instilled after surgical debridement. In this work, we sought to measure in vivo antimicrobial release from PNDJ hydrogels and the antimicrobial concentrations provided in adjacent tissues. PNDJ hydrogels containing tobramycin and vancomycin were administered in four dosing sites in rabbits (intramedullary in the femoral canal, soft tissue defect in the quadriceps, intramuscular injection in the hamstrings, and intra-articular injection in the knee). Gel and tissue were collected up to 72 h after dosing and drug levels were analyzed. In vivo antimicrobial release (43-95% after 72 h) was markedly faster than in vitro release. Drug levels varied significantly depending on the dosing site but not between polymer formulations tested. Notably, total antimicrobial concentrations in adjacent tissue in all dosing sites were sustained at estimated biofilm-eradicating levels for at least 24 h (461-3161 µg/mL at 24 h). These results suggest that antimicrobial-loaded PNDJ hydrogels are promising for improving the treatment of biofilm-based SSIs.

Determination of Tobramycin and Vancomycin Exposure Required to Eradicate Biofilms on Muscle and Bone Tissue In Vitro.

Background: Bacterial biofilms cause chronic orthopaedic infections. Surgical debridement to remove biofilm can be ineffective without adjuvant local antimicrobials because undetected biofilm fragments may remain in the wound and reestablish the infection if untreated. However, the concentrations and duration of antimicrobial exposure necessary to eradicate bacteria from clinical biofilms remain largely undefined. In this study, we determined the minimum biofilm eradication concentration (MBEC) of tobramycin and vancomycin for bacterial biofilms grown on bone and muscle in vitro. Methods: Biofilms of pathogens found in musculoskeletal infections (S. aureus, S. epidermidis, E. faecalis, P. aeruginosa, and E. coli) were established for 72 hr on rabbit muscle and bone specimens in vitro and characterized by SEM imaging and CFU counts. Biofilm-covered tissue specimens were exposed to serial log2 dilutions (4000-31.25 µg/mL) of tobramycin, vancomycin, or a 1:1 combination of both drugs for 6, 24, or 72 hr. Tissues were subcultured following antimicrobial exposure to determine bacterial survival. The breakpoint concentration with no surviving bacteria was defined as the MBEC for each pathogen-antimicrobial-exposure time combination. Results: All tested pathogens formed biofilm on tissue. Tobramycin/vancomycin (1:1) was the most effective antimicrobial regimen with MBEC on muscle (10/10 pathogens) or bone (7/10 pathogens) generally in the range of 100-750 µg/mL with 24 or 72 hr exposure. MBEC decreased with exposure time for 53.3% of biofilms between 6 and 24 hr, 53.3% of biofilms between 24 and 72 hr, and for 76.7% of biofilms between 6 and 72 hr. MBECs on bone were significantly higher than corresponding MBECs on muscle tissue (p < 0.05). In most cases, tissue MBECs were lower compared to previously published MBECs for the same pathogens on polystyrene tissue-culture plates. Conclusions: The majority of MBECs for orthopaedic infections on bone and muscle are on the order of 100-750 µg/mL of vancomycin+tobramycin when sustained for at least 24 hr, which may be clinically achievable using high-dose antimicrobial-loaded bone cement (ALBC).

2018 international consensus meeting on musculoskeletal infection: Summary from the biofilm workgroup and consensus on biofilm related musculoskeletal infections.

Biofilm-associated implant-related bone and joint infections are clinically important due to the extensive morbidity, cost of care and socioeconomic burden that they cause. Research in the field of biofilms has expanded in the past two decades, however, there is still an immense knowledge gap related to many clinical challenges of these biofilm-associated infections. This subject was assigned to the Biofilm Workgroup during the second International Consensus Meeting on Musculoskeletal Infection held in Philadelphia USA (ICM 2018) (https://icmphilly.com). The main objective of the Biofilm Workgroup was to prepare a consensus document based on a review of the literature, prepared responses, discussion, and vote on thirteen biofilm related questions. The Workgroup commenced discussing and refining responses prepared before the meeting on day one using Delphi methodology, followed by a tally of responses using an anonymized voting system on the second day of ICM 2018. The Working group derived consensus on information about biofilms deemed relevant to clinical practice, pertaining to: (1) surface modifications to prevent/inhibit biofilm formation; (2) therapies to prevent and treat biofilm infections; (3) polymicrobial biofilms; (4) diagnostics to detect active and dormant biofilm in patients; (5) methods to establish minimal biofilm eradication concentration for biofilm bacteria; and (6) novel anti-infectives that are effective against biofilm bacteria. It was also noted that biomedical research funding agencies and the pharmaceutical industry should recognize these areas as priorities. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.