Shotgun-metagenomics based prediction of antibiotic resistance and virulence determinants in Staphylococcus aureus from periprosthetic tissue on blood culture bottles

Shotgun-metagenomics may give valuable clinical information beyond the detection of potential pathogen(s). Identification of antimicrobial resistance (AMR), virulence genes and typing directly from clinical samples has been limited due to challenges arising from incomplete genome coverage. We assessed the performance of shotgun-metagenomics on positive blood culture bottles (n = 19) with periprosthetic tissue for typing and prediction of AMR and virulence profiles in Staphylococcus aureus. We used different approaches to determine if sequence data from reads provides more information than from assembled contigs. Only 0.18% of total reads was derived from human DNA. Shotgun-metagenomics results and conventional method results were consistent in detecting S. aureus in all samples. AMR and known periprosthetic joint infection virulence genes were predicted from S. aureus. Mean coverage depth, when predicting AMR genes was 209 ×. Resistance phenotypes could be explained by genes predicted in the sample in most of the cases. The choice of bioinformatic data analysis approach clearly influenced the results, i.e. read-based analysis was more accurate for pathogen identification, while contigs seemed better for AMR profiling. Our study demonstrates high genome coverage and potential for typing and prediction of AMR and virulence profiles in S. aureus from shotgun-metagenomics data.

Revealing Inflammatory Indications Induced by Titanium Alloy Wear Debris in Periprosthetic Tissue by Label-Free Correlative High-Resolution Ion, Electron and Optical Microspectroscopy

The metallic-associated adverse local tissue reactions (ALTR) and events accompanying worn-broken implant materials are still poorly understood on the subcellular and molecular level. Current immunohistochemical techniques lack spatial resolution and chemical sensitivity to investigate causal relations between material and biological response on submicron and even nanoscale. In our study, new insights of titanium alloy debris-tissue interaction were revealed by the implementation of label-free high-resolution correlative microscopy approaches. We have successfully characterized its chemical and biological impact on the periprosthetic tissue obtained at revision surgery of a fractured titanium-alloy modular neck of a patient with hip osteoarthritis. We applied a combination of photon, electron and ion beam micro-spectroscopy techniques, including hybrid optical fluorescence and reflectance micro-spectroscopy, scanning electron microscopy (SEM), Energy-dispersive X-ray Spectroscopy (EDS), helium ion microscopy (HIM) and micro-particle-induced X-ray emission (micro-PIXE). Micron-sized wear debris were found as the main cause of the tissue oxidative stress exhibited through lipopigments accumulation in the nearby lysosome. This may explain the indications of chronic inflammation from prior histologic examination. Furthermore, insights on extensive fretting and corrosion of the debris on nm scale and a quantitative measure of significant Al and V release into the tissue together with hydroxyapatite-like layer formation particularly bound to the regions with the highest Al content were revealed. The functional and structural information obtained at molecular and subcellular level contributes to a better understanding of the macroscopic inflammatory processes observed in the tissue level. The established label-free correlative microscopy approach can efficiently be adopted to study any other clinical cases related to ALTR.

Effects of different tissue specimen pretreatment methods on microbial culture results in the diagnosis of periprosthetic joint infection

Aims Microbiological culture is a key element in the diagnosis of periprosthetic joint infection (PJI). However, cultures of periprosthetic tissue do not have optimal sensitivity. One of the main reasons for this is that microorganisms are not released from the tissues, either due to biofilm formation or intracellular persistence. This study aimed to optimize tissue pretreatment methods in order to improve detection of microorganisms. Methods From December 2017 to September 2019, patients undergoing revision arthroplasty in a single centre due to PJI and aseptic failure (AF) were included, with demographic data and laboratory test results recorded prospectively. Periprosthetic tissue samples were collected intraoperatively and assigned to tissue-mechanical homogenization (T-MH), tissue-manual milling (T-MM), tissue-dithiothreitol (T-DTT) treatment, tissue-sonication (T-S), and tissue-direct culture (T-D). The yield of the microbial cultures was then analyzed. Results A total of 46 patients were enrolled, including 28 patients in the PJI group and 18 patients in the AF group. In the PJI group, 23 cases had positive culture results via T-MH, 22 cases via T-DTT, 20 cases via T-S, 15 cases via T-MM, and 13 cases via T-D. Three cases under ongoing antibiotic treatment remained culture-negative. Five tissue samples provided the optimal yield. Any ongoing antibiotic treatment had a relevant influence on culture sensitivity, except for T-DTT. Conclusion T-MH had the highest sensitivity. Combining T-MH with T-DTT, which requires no special equipment, may effectively improve bacterial detection in PJI. A total of five periprosthetic tissue biopsies should be sampled in revision arthroplasty for optimal detection of PJI. Cite this article: Bone Joint Res 2021;10(2):96–104.

Pathological analysis of periprosthetic soft tissue and modes of failure in revision total joint arthroplasty patients

Objectives: Implant failure leading to revision total joint arthroplasty can occur through a variety of different mechanisms which are typically associated with a soft tissue response adjacent to the implant that provide insight into the underlying etiology of implant failure. The objective of this study was to elucidate mechanisms of implant failure as they relate to histological classification and findings of adjacent periprosthetic tissue. Methods: Histological analysis of soft tissue adjacent to the implant was performed in 99 patients with an average age of 64 years old and grouped into four categories based on the study conducted by Morawietz et al.: Type I (N = 47) Wear particle induced type Type II (N = 7) Infectious type Type III (N = 19) Combined type I and II Type IV (N = 26) Indeterminant type Modes of failure were categorized into five groupings based on the study conducted by Callies et al.: Instability (N = 35), Aseptic Loosening (N = 24), Hardware and/or Mechanical Failure (N = 15), Septic (N = 13), and Other failures (N = 12). We calculated odds ratios and conducted regression analysis to assess the relationship between modes of failure and histological findings as well as modes of failure and comorbidities. Results: Hardware/mechanical failure was independently correlated with histological findings of anucleate protein debris, histiocytes, Staphylococcus epidermidis, and synovitis. Furthermore, hardware/mechanical failure was independently correlated with osteosarcoma as a co-morbidity. Septic failure was associated with histological findings of Enterococcus, granulation tissue, and tissue necrosis as well as comorbidities of Crohn’s disease, deep venous thrombosis, lung disease, and rheumatoid arthritis. Infection was 5.8 times more likely to be associated with Type II histology. Aseptic loosening was associated with histologic findings of synovitis. Conclusion: Our findings support the existing literature on periprosthetic tissue analysis in revision total joint arthroplasty which may improve surgeon understanding of the patholophysiological mechanisms that contribute to implant failure and revision surgery.

324. Implant Sonication Improves Microbiologic Diagnosis of Elbow Prosthetic Joint Infection

Background With a reported incidence of up to 12%, periprosthetic joint infection (PJI) is a frequent complication of total elbow arthroplasty (TEA). Its microbiologic diagnosis is usually based on periprosthetic tissue culture (hereafter referred to as tissue culture), despite the poor sensitivity of this technique. Although implant sonication cultures have been shown to be superior to tissue cultures for hip and knee PJI diagnosis, only a single small study (including fewer than 10 infected implants) has assessed sonication of elbow arthroplasties. Methods We retrospectively analysed 116 sonicate fluid cultures from patients who underwent revision of a TEA at our Institution between 2007 and 2019, comparing results to tissue cultures. Nine elbows who had fewer than 2 tissue samples obtained during surgery were excluded. Using the IDSA guidelines to define PJI, there were 46 infected cases and 61 aseptic failures. We reviewed clinical characteristics and calculated the sensitivity and specificity of periprosthetic tissue culture compared to culture of samples obtained by implant sonication. In addition, we compared the sensitivity of tissue culture to the combination of tissue and sonicate fluid culture. Results A total of 107 elbows were included. Median ages in the aseptic failure and PJI groups were 60 and 67 years, respectively. Gender distribution was similar for both groups (PJI group 62% females; aseptic group 65% females). The most common pathogens were coagulase negative Staphylococcus species (66%), followed by Staphylococcus aureus (18%). The sensitivity of tissue culture was 63% and the sensitivity of sonicate fluid culture was 76% (p=0.14). The specificity of tissue culture was 86% and the specificity of sonicate fluid culture was 100%. Sensitivity of sonicate fluid culture in combination with tissue culture was 91% (p=0.045). Table. Comparison of tests for microbiologic diagnosis of PJI Conclusion The combination of sonicate fluid culture and tissue culture had a greater sensitivity than tissue culture alone for microbiologic diagnosis of elbow TEA infection. Disclosures Joachin Sanchez-Sotelo, M.D, PhD, Elsevier (Other Financial or Material Support, Publishing Royalties)Exactech (Consultant)Oxford Univerity Press (Other Financial or Material Support, Publishing Royalties)Stryker (Grant/Research Support)Wright (Consultant) Robin Patel, MD, Accelerate Diagnostics (Grant/Research Support)CD Diagnostics (Grant/Research Support)Contrafect (Grant/Research Support)Curetis (Consultant)GenMark Diagnostics (Consultant)Heraeus Medical (Consultant)Hutchison Biofilm Medical Solutions (Grant/Research Support)Merck (Grant/Research Support)Next Gen Diagnostics (Consultant)PathoQuest (Consultant)Qvella (Consultant)Samsung (Other Financial or Material Support, Dr. Patel has a patent on Bordetella pertussis/parapertussis PCR issued, a patent on a device/method for sonication with royalties paid by Samsung to Mayo Clinic, and a patent on an anti-biofilm substance issued.)Selux Dx (Consultant)Shionogi (Grant/Research Support)Specific Technologies (Consultant)

2020 John Charnley Award: The antimicrobial potential of bacteriophage-derived lysin in a murine debridement, antibiotics, and implant retention model of prosthetic joint infection

Aims Current treatments of prosthetic joint infection (PJI) are minimally effective against Staphylococcus aureus biofilm. A murine PJI model of debridement, antibiotics, and implant retention (DAIR) was used to test the hypothesis that PlySs2, a bacteriophage-derived lysin, can target S. aureus biofilm and address the unique challenges presented in this periprosthetic environment. Methods The ability of PlySs2 and vancomycin to kill biofilm and colony-forming units (CFUs) on orthopaedic implants were compared using in vitro models. An in vivo murine PJI model of DAIR was used to assess the efficacy of a combination of PlySs2 and vancomycin on periprosthetic bacterial load. Results PlySs2 treatment reduced 99% more CFUs and 75% more biofilm compared with vancomycin in vitro. A combination of PlySs2 and vancomycin in vivo reduced the number of CFUs on the surface of implants by 92% and in the periprosthetic tissue by 88%. Conclusion PlySs2 lysin was able to reduce biofilm, target planktonic bacteria, and work synergistically with vancomycin in our in vitro models. A combination of PlySs2 and vancomycin also reduced bacterial load in periprosthetic tissue and on the surface of implants in a murine model of DAIR treatment for established PJI. Cite this article: Bone Joint J 2020;102-B(7 Supple B):3–10.

Patient-Related Factors Affecting the Sensitivity of Sonication of Removed Hip and Knee Implants Against Conventional Tissue Cultures for Diagnosis of Prosthetic Joint Infections

Despite its decreasing incidence, prosthesis-related infections remain a research, diagnostic, therapeutic and cost-related problem. Our study aim was to compare the diagnostic accuracy of conventional periprosthetic tissue culture and culture of sonication fluid of the explanted hardware and to investigate the role of patientrelated factors affecting the sensitivity of the sonication method. We investigated 70 patients undergoing revision hip or knee arthroplasty, at our institution. Patients’ medical history and demographic characteristics were recorded. We compared the culture of samples obtained by sonication of explanted hip and knee prostheses with conventional culture of periprosthetic tissue for the microbiological diagnosis of prosthetic-joint infection. Thirty-two patients had septic loosening and 38 aseptic loosening (48 hip prostheses and 22 knee prostheses). The sensitivities of sonication fluid culture and conventional tissue cultures were 81.25% and 56.25%, respectively (p-value = 0.043). The sensitivity of the sonication method was statistically higher in obese, diabetic patients, with age above 60, in uncemented arthroplasties and in arthroplasties because of primary osteoarthritis (p-values < 0.05). The sonication method has a greater sensitivity than the conventional periprosthetic tissue cultures for the periprosthetic infections, especially in obese, diabetic patients, with age above 60, in uncemented arthroplasties and in arthroplasties because of primary osteoarthritis.