Aim. Diagnosing Orthopaedic infection is limited by the sensitivity of culture methods. Next generation sequencing (NGS) offers an alternative approach for detection of microorganisms from clinical specimens. However, the low ratio of pathogen DNA to human DNA often inhibits detection of microorganisms from specimens. Depletion of human DNA may enhance the detection of microbial DNA. 1. Our aim was to compare four DNA extraction methods for the recovery of microbial DNA from orthopaedic samples for NGS. Method. Simulated samples; pooled culture negative sample matrix was spiked with known concentrations of microorganisms, each panel consisting of 7 samples. Broth culture was performed on simulated samples for comparison with NGS. *. . DNA Extraction; total nucleic acid extraction was performed on an automated extraction platform. **. using the viral NA assay. Modifications included: (1) mechanical lysis (glass beads), (2) lysis of human cells (saponin 0.025%), turbo DNase treatment and (3) mechanical lysis and addition of MspJI enzyme post-extraction for methylated DNA digestion. Detection of human and microbial DNA; human endogenous (HE) gene rtPCR. ***. was utilised following manufacturer's recommendations. Microbial DNA was detected using SYBR green 16s ribosomal RNA rtPCR with high resolution melt-curve analysis. ****. . Results. Broth culture recovered 64% (9/14) of the microorganisms from simulated samples. A significant increase (p<0.01) in the cycle threshold (C. T. ) (median C. T. 25.9 IQR 25.5, 26.1) of the HE gene rtPCR was observed using extraction method b, indicating a significant reduction in human DNA. No significant change (p=0.38) in the C. T. of the HE gene rtPCR was observed between the baseline method (median C. T. 19.2 IQR 18.5, 19.7) and modifications a (median C. T. 18.4 IQR 18.2, 19.4) and c (median C. T. 19.3 IQR 18.6, 19.4). Detection of microbial DNA was successful using the base line extraction method and modification a. Microbial DNA was not detected using the 16s ribosomal RNA rtPCR for modifications b and c. Conclusions. This study has demonstrated that modification of DNA extraction methods using selective enzymatic digestion of human DNA negatively impacts on the recovery of microbial DNA from simulated specimens. Total DNA extraction allows the successful recovery of microbial DNA alongside a significant amount of human DNA. The effect of the presence of human DNA will be subsequently assessed through NGS CosmosID analysis to establish if NGS is more sensitive than broth based culture

Aims

Bacterial infection activates neutrophils to release neutrophil extracellular traps (NETs) in bacterial biofilms of periprosthetic joint infections (PJIs). The aim of this study was to evaluate the increase in NET activation and release (NETosis) and haemostasis markers in the plasma of patients with PJI, to evaluate whether such plasma induces the activation of neutrophils, to ascertain whether increased NETosis is also mediated by reduced DNaseI activity, to explore novel therapeutic interventions for NETosis in PJI in vitro, and to evaluate the potential diagnostic use of these markers.

Non-invasive sampling of tumor-derived genetic material in circulation through liquid biopsy may be very beneficial for an accurate diagnosis and evaluation of response to treatment in patients with malignant and benign soft tissue tumors. We previously showed that tumor-derived genomic aberrations can be detected in plasma of patients with leiomyosarcoma (LMS) and leiomyoma (LM). In LMS patients, we also showed that the levels of circulating tumor DNA (ctDNA) correspond with response to treatment. We developed an approach tailored to genomic profile of LMS (characterized by intermediate levels of point mutations and copy number alterations, CNAs). Based on TCGA data, we designed a panel of 89 most frequently mutated genes in LMS, which we profiled in plasma DNA by deep sequencing. In parallel, plasma samples were analyzed by shallow whole genome sequencing for detection of CNAs. With this approach, we detected ctDNA in 71% (20/28) of samples from 6/7 patients with advanced disease with >98% specificity. The combination approach for orthogonal profiling of point mutations and CNAs proved to increase the sensitivity of ctDNA detection. Currently, we seek to further improve the sensitivity of ctDNA detection by refining our capture panel and tracking LMS-specific DNA methylation markers in circulation, in addition to point mutations and CNAs. The ultimate goals of our ctDNA studies are 1) to develop a highly sensitive assay for evaluation of response to therapy and long-term surveillance for patients with LMS, and 2) to develop a blood-based test for accurate pre-operative distinction between LMS and LM. To identify LMS-specific DNA methylation markers, we analyzed a test cohort of 76 LM, 35 uterine LMS and 31 extra-uterine LMS by Illumina Infinium EPIC arrays. We identified differentially methylated CpGs between LM and uterine LMS, and between LM and all LMS using a newly developed custom pipeline in R. The results of this analysis are currently being validated in a new dataset of 41 LM and 153 LMS generated by our group. Recently published (PMID: 34301934) genomic data from new 53 LMS samples are used to refine the panel of the most frequently mutated genes that we identified previously in the LMS TCGA data. Our preliminary analysis of test cohort revealed >270 differentially methylated CpGs between LM and uterine LMS, and >1000 differentially methylated CpGs between LM and all LMS. The preliminary analysis of genomic data shows that the initial panel of 89 frequently mutated genes could be substantially narrowed down to cover only selected tumor suppressor genes. Once validated, these results will be used to refine the ctDNA assay for LMS and LM. Our results point to multiple epigenetic markers that could be used for ctDNA profiling, in addition to point mutations or CNAs. Further validation will allow us to select the most reliable LMS- and LM-specific DNA methylation markers and the most frequently mutated regions across independent datasets, and these markers will be incorporated into our new ctDNA test for a concurrent detection of point mutations, CNAs and DNA methylation markers in circulation

Specific and rapid detection methods for spinal tuberculosis, with sufficient sensitivity in HIV-1 co-infected individuals, are needed, to ensure early initiation of appropriate treatment to prevent physical disability and neurological fallout. In addition, understanding the systemic and local pathophysiology of spinal tuberculosis, and its interaction with HIV-1 infection, is crucial to guide future therapeutic interventions. We prospectively enrolled adult patients presenting with signs and symptoms of suspected spinal tuberculosis, at Groote Schuur Hospital, between November 2020 and December 2021. TB diagnostic testing was performed on open and CT-guided spinal biopsies using Xpert MTB/RIF Ultra compared to gold standards TB culture and histology. A highly sensitive droplet digital PCR assay for detecting and quantifying Mycobacterium tuberculosis complex (MTBC) and HIV-1 DNA was tested. Plasma inflammatory proteins were measured to assess systemic inflammation. Xpert Ultra had a high sensitivity of 94.7% and specificity of 100% for STB against TB culture and histology in both open and CT-guided biopsy samples. The ddPCR assay confirmed TB detection in 94% of patients with positive Xpert Ultra results. Four patients with negative TB diagnostic results had MTBC DNA detected by ddPCR. HIV-1 DNA was detected in the spinal tissues from all HIV-1-infected patients. MTBC DNA levels were significantly higher in HIV-1-co-infected spinal tissue samples (p< 0.01). We identified four biomarkers significantly associated with higher bacterial burden at the disease site (p< 0.01). Xpert Ultra and MTBC ddPCR improve the detection of STB. DdPCR can be utilized as an additional, highly sensitive tool for detecting and quantifying Mtb, in pathological samples that may be paucibacillary. These findings provide novel diagnostic and pathophysiologic insight into STB, in the context of HIV-1 infection, and provide rationale to include these tests in hospital and research settings for patients from communities burdened by TB and HIV-1

Aim. Metagenomic nanopore sequencing is demonstrating potential as a tool for diagnosis of infections directly from clinical samples. We have previously shown nanopore sequencing can be used to determine the causative bacterial species in prosthetic joint infections (PJI). However, to make predictions regarding antimicrobial resistance, human DNA contamination must be reduced so a greater proportion of sequence data corresponds to the microbial portion of the DNA extract. Here, we utilise selective DNA extraction from sonication fluid samples to begin to make predictions regarding antimicrobial resistance in PJI. Method. We investigated host cell DNA depletion with 5% saponin selective human cell lysis followed by nuclease digestion. Subsequently, bacterial cells were mechanically lysed before DNA extraction. Sequencing libraries from samples treated with and without saponin were prepared with a Rapid PCR Barcoding Kit. 1. and sequenced in multiplexes of 2–8 samples/flowcell on a GridION. Sequencing reads were analysed using the CRuMPIT pipeline and thresholds to indicate presence of a specific bacterial genus/species were investigated. Antimicrobial resistance determinants were detected using previously published sequences specifically for Staphylococcus aureus, as an example organism frequently causing PJI. Results. 247 DNA extracts from 115 sonication fluids plus controls were subjected to metagenomic sequencing, comprising extracts from 67 culture-positive (10 of which were culture-positive at <50 CFU/ml) and 48 culture-negative samples. 5% saponin depleted human DNA contamination, reducing the number of human sequenced bases to a median 12% from 98% in comparison to 5μm filtration without saponin. In 11 samples 5% saponin depleted human bases by <12% in comparison to 5μm filtration, which may be indicative of incomplete depletion. Bacteria observed in sonication fluid culture were identified to species-level in 49/65 (75%) cases, and to genus-level in 51/65 (78%). Specificity of sequencing was 103/114 (90%). Sequencing made a completely successful prediction of antimicrobial susceptibility in 8/19 S. aureus culture-positive samples treated with 5% saponin, and a partial prediction in 5/19 for the 8 antibiotics investigated. Without 5% saponin treatment sequencing could only detect a limited number of AMR determinants in 3/19 samples. Sequencing correctly predicted 13/15 (87%) resistant and 74/74 (100%) susceptible phenotypes where sufficient sequence data were available. Conclusions. Nanopore metagenomic sequencing can provide species identification in PJI. Additionally, depletion of human DNA improves depth of coverage and allows detection of antimicrobial resistance determinants, demonstrating as a proof of principle that nanopore sequencing could potentially provide a complete diagnostic tool in PJI. 1. Oxford Nanopore Technologies

A novel injectable hydrogel based on DNA and silicate nanodisks was fabricated and optimized to obtain a suitable drug delivery platform for biomedical applications. Precisely, the hydrogel was designed by combining two different type of networks: a first network (type A) made of interconnections between neighboring DNA strands and a second one (type B) consisting of electrostatic interactions between the silicate nanodisks and the DNA backbone. The silicate nanodisks were introduced to increase the viscosity of the DNA physical hydrogel and improve their shear-thinning properties. Additionally, the silicate nanodisks were selected to modulate the release capability of the designed network. DNA 4% solutions were heated at 90°C for 45 seconds and cooled down at 37°C degree for two hours. In the second step, the silicate nanodisks suspension in water at different concentrations (0.1 up to 0.5%) were then mixed with the pre-gel DNA hydrogels to obtain the nanocomposite hydrogels. Rheological studies were carried out to investigate the shear thinning properties of the hydrogels. Additionally, the hydrogels were characterized by scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR), and X-ray photoelectron microscopy. The hydrogels were loaded with the osteoinductive drug dexamethasone and its release was tested in vitro in phosphate buffer pH 7.4. The drug activity upon release was tested evaluating the osteogenic differentiation of human adipose derived stem cells (hASCs) in vitro through analysis of main osteogenic markers and quantification of alkaline phosphatase activity and calcium deposition. Finally, the hydrogels were tested in vivo and injected into cranial defects in rats to assess their biocompatibility and bone regeneration potential. The inclusion of the silicate nanodisks increased the viscosity of the hydrogels and the best results were obtained with the highest concentration of the nanoclay (0.5%). The hydrogels possessed shear-thinning properties as demonstrated by cyclic strain sweep tests and were able to recover their original storage modulus G' upon removal of strain. Such improvement in the injectable properties of the formulated hydrogels was mainly attributed to the formation of electrostatic interactions between the silicate nanodisks and the phosphate groups of the DNA backbone as confirmed by XPS analysis of the O, N, and P spectra. Additionally, laponite was able to sustain the release of the osteoinductive drug dexamethasone which was instead completely released from the DNA-based hydrogels after a week. The drug after being released was still active and promoted the osteogenic differentiation of hASCs as confirmed by ALP expression and expression of main osteogenic markers including ALP and COLA1. Finally, the gels proved to be biocompatible in vivo when injected into cranial defects and promoted bone formation at the periphery of the defect after a month post-treatment. A novel injectable shear-thinning DNA-based hydrogel was characterized and tested for its drug delivery properties. The hydrogel can promote the sustain release of a small molecule like dexamethasone and be biocompatible in vitro and in vivo. Due to these promising findings, the designed system could find also applicability for the delivery of growth factors or other therapeutic molecules

Aim. The clinical relevance of microbial DNA detected via next-generation sequencing (NGS) remains unknown. This multicenter study was conceived to: 1) identify species on NGS that may predict periprosthetic joint infection (PJI), then 2) build a predictive model for PJI in a developmental cohort, and 3) validate predictive utility of the model in a separate multi-institutional cohort. Method. Fifteen institutions prospectively collected samples from 194 revision TKA and 184 revision THA between 2017–2019. Synovial fluid, tissue and swabs were obtained intraoperatively and sent to MicrogenDx (Lubbock, TX) for NGS analysis. Reimplantations were excluded. Patients were classified per the 2018 ICM definition of PJI. DNA analysis of community similarities (ANCOM) was used to identify 17 bacterial species of 294 (W-value>50) for differentiating infected vs. noninfected cases. Logistic regression with LASSO selection and random-forest algorithms were then used to build a model for predicting PJI. ICM classification was the response variable (gold-standard) and species identified through ANCOM were predictors. Patients were randomly allocated 1:1 into training and validation sets. Using the training set, a model for PJI diagnosis was generated. The entire model-building procedure and validation was iterated 1000 times. Results. The model's assignment accuracy was 75.9%. There was high accuracy in true-negative and false-negative classification using this model, which has previously been a criticism of NGS. Specificity was 97.1%, PPV 75.0% and NPV 76.2%. On comparison of abundance between ICM-positive and ICM-negative patients, Staphylococcus aureus was the strongest contributor (F=0.99) to model predictive power. In contrast, Cutibacterium acnes was less predictive (F=0.309) and abundant across infected and noninfected revisions. Discussion. This is the first study to utilize predictive algorithms on a large multicenter dataset to transform analytic NGS data into a clinically relevant diagnostic model. Our collaborative findings suggest NGS may be an independent adjunct for PJI diagnosis, while also facilitating pathogen identification. Future work applying machine-learning will improve accuracy and utility of NGS

Aim. Cutibacterium acnes (C. acnes) is the most cultured organism implicated in periprosthetic shoulder infections. Nevertheless, the clinical significance of its persistence on the skin surface and in the deep layers during shoulder arthroplasty surgery remains still unknown. The purpose of this study was to know if the C. acnes isolate present in deep tissues at the end of a primary shoulder arthroplasty could be responsible for shoulder arthroplasty infection. Method. Prospective study including 156 patients undergoing primary shoulder arthroplasty. In all the patients included 5 to 12 tissue samples were obtained and were specifically cultured to detect C. acnes presence. DNA was extracted from the C. acnes colonies selected with the QIAsymphony DSP Virus/Pathogen Midi Kit (Qiagen, Hilden, Germany). Libraries were prepared using Nextera XT kit (Illumina) and sequenced in an Illumina MiSeq sequencer. Sequencing files were pre-processed using The Microbial Genome Atlas pipeline. Samples that failed on QC analysis were discarded for further analysis. Isolate nucleotide distances were calculated using Genome-based distance matrix calculator from the enveomics collection. Comparative genomic analysis was performed between intra- and inter-patients’ isolates. Data analysis was performed using R 3.6.3. Results. For twenty-seven out of 156 patients (17.31%), C. acnes was present at the end of the primary surgery. Two of these patients (both male) developed a C. acnes periprosthetic shoulder infection after 6 and 4 months from the primary surgery. DNA from the C. acnes responsible for the periprosthetic infection was further analysed by whole genome sequencing (WGS). Average Nucleotide Identity (ANI) value was assessed, measuring the nucleotide-level genomic similarity between genome pairs. We found a clear ANI clustering in two major groups which corresponded, mainly, to the associated phylotype (97%–98% ANI). Moreover, when analysing both isolates that developed a periprosthetic shoulder infection, we found that all the revision-surgery isolates clustered nearer to their corresponding primary-surgery isolates (99.4% of similarity) than to the other independent bacterial isolates, supporting the causal relationship between the initial and the delayed infection. Conclusions. C. acnes present at the end of the primary surgery can be the cause of early- or delayed-periprosthetic joint infections in shoulder arthroplasty, revealing the potential route of infection. Therefore, efforts must be made in terms of antibiotic prophylaxis and skin preparation to limit infections of total shoulder arthroplasties

Background. The identification of novel biomarker which is highly specific and sensitive for periprosthetic joint (PJI) have the potential to improve diagnostic accuracy and ultimately improve patient outcomes. Thus, the aim of this systemic review is to identify and evaluate novel biomarkers for the preoperative diagnostics of PJI. Methods. MEDLINE, EMBASE, PubMed and Cochrane Library databases identified from 1. st. of January 2018 to 30. th. of September. 2022. We used “periprosthetic joint infection” OR “prosthetic joint infection” OR “periprosthetic infection” as the diagnosis of interest and the target index applied AND “marker”. To focus on novel biomarkers already used biomarkers of the established PJI diagnostic criteria of MSIS, ICM and EBJIS were not included in the analysis. These three criteria were considered the reference standard during quality assessment. Results. A total of 19 studies were included. In these, fourteen different novel biomarkers were analyzed. Fifteen studies (79%) had prospective designs and the other four (22%) were retrospective studies. Six studies (33%) included only periprosthetic knee infections and thirteen (67%) included periprosthetic knee and hip infections. Proteins were analyzed in most cases (nine studies), followed by molecules (three studies), exosome (two studies) as well as DNA (two studies), interleukin (one study) and lysosome (one study). One novel and promising marker that had been frequently analyzed is calprotectin. Conclusion. No marker demonstrated higher sensitivity and specificity than already known parameters used for standardized treatment based on established PJI definitions. Further studies are needed to elucidate the benefit and usefulness of implementing new biomarkers in diagnostic PJI settings

Aim. While 16S rRNA PCR - Sanger sequencing has paved the way for the diagnosis of culture-negative bacterial infections, it does not provide the composition of polymicrobial infections. We aimed to evaluate the performance of the Nanopore-based 16S rRNA metagenomic approach using partial-length amplification of the gene, and to explore its feasibility and suitability as a routine diagnostic tool for bone and joint infections (BJI) in a clinical laboratory. Method. Sixty-two clinical samples from patients with BJI were sequenced on MinION* using the in-house partial amplification of the 16S rRNA gene. BJI were defined based on the ICM Philly 2018 and EBJIS 2021 criteria. Among the 62 samples, 16 (26%) were culture-positive, including 6 polymicrobial infections, and 46 (74%) were culture-negative from mono- and polymicrobial infections based on Sanger-sequencing. Contamination, background noise definition, bacterial identification, and time-effectiveness issues were addressed. Results. Results were obtained within one day. Setting a threshold at 1% of total reads overcame the background noise issue and eased interpretation of clinical samples. The partial 16S rRNA metagenomics approach had a greater sensitivity compared both to the culture method and the Sanger sequencing. All the 16 culture-positive samples were confirmed with the metagenomic sequencing. Bacterial DNA was detected in 32 culture-negative samples (70%), with pathogens consistent with BJI. The 14 Nanopore negative samples included 7 negative results confirmed after implementation of other molecular techniques and 7 false-negative MinION results: 3 Kingella kingae infections detected after targeted-PCR only, 2 Staphylococcus aureus infections and 2 Pseudomonas aeruginosa infections sterile on agar plate media and detected only after implementation of blood culture media, advocating for the very low inoculum. Conclusions. The results discriminated polymicrobial samples, and gave accurate bacterial identifications compared to Sanger-based results. They confirmed that Nanopore technology is user-friendly as well as cost- and time-effective. They also indicated that 16S rRNA targeted metagenomics is a suitable approach to be implemented for routine diagnosis of culture-negative samples in clinical laboratories. * Oxford Nanopore Technologies

Osteoarthritis (OA) is the most common form of arthritis and one of the ten most disabling diseases in developed countries. Total joint replacement (TJR) is considered by far as the most effective treatment for end-stage OA patients. The majority of patients achieve symptomatic improvement following TJR. However, about 22% of the TJR patients either do not improve or deteriorate after surgery. Several potential non-genetic predictors for the TJR outcome have been investigated. However, the results were either inconclusive or had very limited predictive power. The aim of this study was to identify genetic variants for the poor outcome of TJR in primary OA patients by a genome-wide association study (GWAS). Study participants were total knee or hip replacement patients due to primary OA who were recruited to the Newfoundland Osteoarthritis Study (NFOAS) before 2017. The Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) was used to assess pain and functional impairment pre- and 3.99±1.38 years post-surgery. Two non-responder classification criteria were used in our study. One was defined by an absolute WOMAC change score. Participants with a change score less than 7/20 points for pain were considered as pain non-responders; and those with less than 22/68 points for function were classified as function non-responders. The second one was the Outcome Measures in Arthritis Clinical Trials and the Osteoarthritis Research Society International (OMERACT-OARSI) criteria. Blood DNA samples were genotyped using the Illumina GWAS microarrays genotyping platform. The quality control (QC) filtering was performed on GWAS data before the association of the genetic variants with non-responders to TJR was tested using the GenABEL package in R with adjustment for the relatedness of the study population and using the commonly accepted GWAS significance threshold p < 5*10. −8. to control multiple testing. In total, 316 knee and 122 hip OA patients (mean age 65.45±7.62 years, and 58% females) passed the QC check. These study participants included 368 responders and 56 non-responders to pain, and 364 responders and 68 non-responders to function based on the absolute WOMAC point score change classification. While 377 responders and 56 non-responders to pain, and 366 responders and 71 non-responders to function were identified by the OMERACT-OARSI classification criteria. Interestingly, the same results were obtained by both classification methods, and we found that the G allele of rs4797006 was significantly associated with pain non-responders with odds ratio (OR) of 5.12 (p<7.27×10. -10. ). This SNP is in intron one of the melanocortin receptor 5 (MC5R) gene on chr18. This gene plays central roles in immune response, pain sensitivity, and negative regulation of inflammatory response to antigenic stimulus. The A allele of rs200752023 was associated with function non-responders with OR of 4.41 (p<3.29×10. -8. ). The SNP is located in intron three of the RNA Binding Fox-1 Homolog 3 (RBFOX3) gene on chr17 which has been associated with numerous neurological disorders. Our data suggested that two chromosomal regions are associated with TJR poor outcomes and could be the novel targets for developing strategies to improve the outcome of the TJR

Total joint replacement (TJR) is by far the most effective therapy for end-stage OA patients. Most of patients achieve joint pain reduction and function improvement following to TJR, however up to 22% of them either do not improve or deteriorate after surgery. The aim of this study was to identify genetic variants to be associated with poor outcome of TJR in primary OA patients by a genome-wide association approach (GWAS). Study participants were primary OA patients from the Newfoundland Osteoarthritis Study (NFOAS) that comprised total knee or hip replacement and recruited before 2016 in St. John's, NL. DNA samples were extracted from patients' blood. Study participants completed their pre-operation and 3.99±1.38 years post-surgery outcome assessment using the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC). DNA samples were genotyped using the genome-wide Illumina HumanOmni2.58 genotyping microarray containing 2.4 million SNPs. Pre-association quality control filtering was conducted for the raw genotyping data using PLINK 1.7 program, and genotype imputation was performed using the IMPUTE2 algorithm with multiple population reference data from 1000 Genome Project. The imputed data with ∼3.1 million variants was used to test the association with non-responders to TJR using the additive genetic model. Eighty three primary OA patients (44 responders and 39 non-responders) were included in the analysis. Association analysis detected three chromosomal regions on chr5, 7, and 8 to be significantly associated with non-responding to pain. The top SNPs at these loci are intergenic variants that include SNP (rs17118094, p=4.4×10-5) on chr5. This SNP is adjacent to SGCD gene that plays an important role in muscular strength and maintenance. Another associated SNP (rs71572810, p=4.7×10-5) is nearby IMMP2L gene on chr7. This gene is reported to be associated with behavioral abnormalities. Finally, SNP (rs6992938, p=5.8×10-5) on chr8 is located downstream of TRPA1 gene that is known to have a central role in the pain response to endogenous inflammatory mediators. Three loci were also found to be significantly associated with non-responding to function. The lead variant in the locus on chr1 is an intergenic SNP (rs9729377, p=1.7×10-5) falling between CTBS and MCOLN2 genes. CTBS gene is associated with TNF-α, a cytokine that stimulate the inflammation acute phase reaction, and MCOLN2 gene plays a role in the chemokine secretion and macrophage migration in the innate immune response. Other top SNPs in loci on chr2 and 10 harbor CCDC93, INSIG2, and KLF6 genes that are associated with heel bone mineral density, hypercholesterolemia, obesity and BMI. To our knowledge, this project is the first study that investigated the association between genetic factors and TJR non-responders. Our results demonstrated that genes related to muscle strength, behavioral trait, pain response, and inflammation play a significant role in poor outcome of TJR, warranting further investigation

Aim. Culture of multiple periprosthetic tissue samples is the current gold-standard for microbiological diagnosis of prosthetic joint infections (PJI). Additional diagnostic information may be obtained through sonication fluid culture of explants. These current techniques can have relatively low sensitivity, with prior antimicrobial therapy or infection by fastidious organisms particularly influencing culture results. Metagenomic sequencing has demonstrated potential as a tool for diagnosis of bacterial, viral and parasitic infections directly from clinical samples, without the need for an initial culture step. We assessed whether metagenomic sequencing of DNA extracts from sonication fluid can provide a sensitive tool for diagnosis of PJI compared to sonication fluid culture. Method. We compared metagenomic sequencing with standard aerobic and anaerobic culture in 97 sonication fluid samples from prosthetic joint and other orthopaedic device-related infections. Sonication fluids were filtered to remove whole human cells and tissue debris, then bacterial cells were mechanically lysed before DNA extraction. DNA was sequenced and sequencing reads were taxonomically classified using Kraken. Using 50 derivation samples, we determined optimal thresholds for the number and proportion of bacterial reads required to identify an infection and confirmed our findings in 47 independent validation samples. Results. A total of 131 sonication fluids were aerobically and anaerobically cultured and underwent metagenomic sequencing. From the first 72 sonication fluid samples sequenced 22 samples from six batches were excluded, as these samples and negative controls from the same batches showed similar contamination. The remaining 50 samples, the derivation set, were used to determine optimal sequence thresholds for identifying true infection. Of 59 subsequently sequenced validation samples, 12 from a single batch were excluded as the negative control was contaminated with Propionibacterium acnes, leaving 47 validation samples. Compared to sonication fluid culture, the species-level sensitivity of metagenomic sequencing was 61/69(88%,95%CI 77–94%)(derivation samples 35/38[92%,79–98%]; validation samples 26/31[84%,66–95%]), and genus-level sensitivity was 64/69(93%,84–98%). Species-level specificity, adjusting for plausible fastidious causes of infection, species found in concurrently obtained tissue samples, and prior antibiotics, was 85/97(88%,79–93%)(derivation 43/50[86%,73–94%], validation 42/47[89%,77–96%]). High levels of human DNA contamination were seen despite use of laboratory methods to remove it. Conclusions. We demonstrate as a proof of principle that metagenomic sequencing can provide accurate diagnostic information in PJI. Further depletion of human DNA will lead to improved genomic information on the cause of infection, strengthening the case for metagenomic sequencing as a diagnostic tool in PJI

Aim. Periprosthetic joint infection is an increasing reason for revision surgery. Tissue cultures are a standard (std.) diagnostic procedure but may be hindered by bacteria that are difficult to cultivate. The use of dithiothreitol (DTT) to detach the formed biofilm has been proposed to improve the diagnostic security. The aim was to compare the diagnosis results using the microDTTect device with the routine PJI diagnostics and next generation sequencing (NGS) from DTT treated explants. Method. 66 patients with revision surgeries were included in this study (38 aseptic; 28 septic). We compared std. microbiology tissue cultures with the microDTTect cultures of the DTT treated explants and NGS of bacterial DNA isolated from DTT solution. Results. In 75% of the septic cases, the std. microbiology was in line with the microDTTect cultures. In 8% of the aseptic cases, the microDTTect culture indicated a present pathogen. In 71% of the septic cases, NGS was compared to the std. microbiology and NGS. The concordance in the aseptic cohort between NGS and std. microbiology was 79%. Staphylococcus were most frequently detected by all three techniques Polymicrobial infections, were detected less frequently by culturing techniques, but with a high sensitivity using NGS. Conclusion. Our data indicate that tissue cultures show a similar reliability compared to the other techniques. The DTT culture method had a sensitivity of 75% while the specificity was 92%. NGS had a sensitivity of 71% and a specificity of 79%. These results may improve the treatment decision in clinical practice

Abstract. Background. The COVID-19 pandemic has strongly impacted elective orthopaedic surgery. At our trust, a geographically discrete elective site deals with planned orthopaedic surgery. There was a need to define a green pathway to deliver surgical care safely and efficiently, and tackle mounting waiting lists. Methods. Records of patients operated at our elective site, between 1. st. July 2020 and 14. th. January 2021, under a green pathway, including pre-operative self-isolation, COVID screening and segregating perioperative patients, were reviewed, and analysed retrospectively. Patients who did not attend (DNA) their post-operative follow-up appointments were identified. Finally, regional COVID incidence was compared with that in our centre. Results. During this period, 2466 patients were admitted for elective orthopaedic surgery, of which sixteen (0.6%) tested COVID-positive. Among these, two tested positive during asymptomatic in-patient screening, five tested positive within two weeks of discharge, while six tested positive beyond two weeks. One patient tested positive on the day of surgery, which was then postponed. Fourteen (87.5%) patients who tested positive recovered, with two COVID-related mortalities. We identified 34 (1.4%) DNA patients. The COVID incidence in our centre closely paralleled the regional incidence. There were no major COVID outbreaks and no definite evidence of in-hospital transmission. Conclusion. This green pathway has helped streamline patient flow for continuing elective surgery safely through the pandemic, under cautionary monitoring. Modulating elective activity in response to regional COVID rates is vital for addressing waiting lists. Given the encouraging results, this pathway is an effective measure for maintaining elective activity through the pandemic

Intervertebral disc (IVD) degeneration plays a major role in low back pain which is the leading cause of disability. Current treatments in severe cases require surgical intervention often leading to adjacent segment degeneration. Injectable hydrogels have received much attention in recent years as scaffolds for seeding cells to replenish disc cellularity and restore disc properties and function. However, they generally present poor mechanical properties. In this study, we investigated several novel thermosensitive chitosan hydrogels for their ability to mimic the mechanical properties of the nucleus pulposus (NP) while being able to sustain the viability of NP cells, and retain proteoglycans. CH hydrogels were prepared by mixing the acidic chitosan solution (2% w/v) with various combinations of three gelling agents: sodium hydrogen carbonate (SHC) and/or beta-glycerophosphate (BGP) and/or phosphate buffer (PB) (either BGP0.4M, SHC0.075M-BGP0.1M, SHC0.075M-PB0.02M or SHC0.075M-PB0.04M). The gelation speed was assessed by following rheological properties within 1h at 37°C (strain 5% and 1Hz). The mechanical properties were characterized and compared with that of human NP tissues. Elastic properties of the hydrogels were studied by evaluating the secant modulus in unconfined compression. Equilibrium modulus was also measured, using an incremental stress-relaxation test 24h after gelation in unconfined compression (5% strain at 5%/s followed by 5min relaxation, five steps). Cells from bovine IVD were encapsulated in CH-based gels and maintained in culture for 14 days. Cytocompatibility was assessed by measuring cell viability, metabolism and DNA content. Glycosaminoglycan (GAG) synthesis (retained in the gel and released) was determined using DMMB assay. Finally injectability was tested using human cadaveric discs. Unconfined compression confirmed drastically enhanced mechanical properties compared to conventional CH-BGP hydrogels (secant Young modulus of 105 kPa for SHC0.075PB0.02 versus 3–6 kPa for BGP0.04). More importantly, SHC0.075PB0.02 and SHC0.075BGP0.1 hydrogels exhibited mechanical properties very similar to NP tissue. For instance, equilibrium modulus was 5.2±0.6 KPa for SHC0.075PB0.02 and 8±0.8 KPa for SHC0.075BGP0.1 compared to 6.1±1.7 KPa for human NP tissue. Rheological properties and gelation time (G′=G″ after less than 15 s at 37°C, and rapid increase of G') of these hydrogels also appear to be adapted to this application. Cell survival was greater than 80% in SHC0.075BGP0.1 and SHC0.075PB0.02 hydrogels. Cells encapsulated in the new formulations also showed significantly higher metabolic activity and DNA content after 14 days of incubation compared to cells encapsulated in BGP0.4 hydrogel. Cells encapsulated in SHC0.075BGP0.1 and SHC0.075PB0.02 produced significantly higher amounts of glycosaminoglycans (GAG) compared to cells encapsulated in SHC0.075PB0.04 and BGP0.4 hydrogels. The total amount of GAG was higher in SHC0.075BGP0.1 hydrogel compared to SHC0.075PB0.02. Interestingly, both the SHC0.075BGP0.1 and SHC0.075PB0.02 hydrogels retained similar amounts of GAG. Injectability through a 25G syringe, filling of nuclear clefts and good retention in human degenerated discs was demonstrated for SHC0.075PB0.02 hydrogel. SHC0.075BGP0.1 appears to be a particularly promising injectable scaffold for IVD repair by providing suitable structural environment for cell survival, ECM production and mechanical properties very similar to that of NP tissue

Currently, different techniques to evaluate the biocompatibility of orthopaedic materials, including two-dimensional (2D) cell culture for metal/ceramic wear debris and floating 2D surfaces or three-dimensional (3D) agarose gels for UHMWPE wear debris, are used. Moreover, cell culture systems evaluate the biological responses of cells to a biomaterial as the combined effect of both particles and ions. We have developed a novel cell culture system suitable for testing the all three type of particles and ions, separately. The method was tested by evaluating the biological responses of human peripheral blood mononuclear cells (PBMNCs) to UHMWPE, cobalt-chromium alloy (CoCr), and Ti64 alloy wear particles. Methods. Clinically relevant sterile UHMWPE, CoCr, and Ti64 wear particles were generated in a pin-on-plate wear simulator. Whole peripheral blood was collected from healthy human donors (ethics approval BIOSCI 10–108, University of Leeds). The PBMNCs were isolated using Lymphoprep (Stemcell, UK) and seeded into the wells of 96-well and 384-well cell culture plates. The plates were then incubated for 24 h in 5% (v/v) CO. 2. at 37°C to allow the attachment of mononuclear phagocytes. Adherent phagocytes were incubated with UHMWPE and CoCr wear debris at volumetric concentrations of 0.5 to 100 µm. 3. particles per cell for 24 h in 5% (v/v) CO. 2. at 37°C. During the incubation of cells with particles, for each assay, two identical plates were set up in two configurations (one upright and one inverted). After incubation, cell viability was measured using the ATPlite assay (Perkin Elmer, UK). Intracellular oxidative stress was measured using the DCFDA-based reactive oxygen species detection assay (Abcam, UK). TNF-α cytokine was measured using sandwich ELISA. DNA damage was measured by alkaline comet assay. The results were expressed as mean ± 95% confidence limits and the data was analysed using one-way ANOVA and Tukey-Kramer post-hoc analysis. Results and Discussion. Cellular uptake of UHMWPE, CoCr and Ti64 particles was confirmed by optical microscopy. PBMNCs incubated with UHMWPE particles did not show any adverse responses except the release of significant levels of TNF-α cytokine at 100 µm. 3. particles per cell, when in contact with particles. PBMNCs incubated with CoCr wear particles showed adverse responses at high particle doses (100 µm. 3. particles per cell) for all the assays. Moreover, cytotoxicity was observed to be a combined effect of both particles and ions, whereas oxidative stress and DNA damage were mostly caused by ions. Ti64 wear particles did not show any adverse responses except cytotoxicity at high particle doses (100 µm. 3. particles per cell). Moreover, this cytotoxicity was mostly found to be a particle effect. In conclusion, the novel cell culture system is suitable for evaluating the biological impact of orthopaedic wear particles and ions, separately

Propionibacterium acnes infection of the shoulder after arthroplasty is a common complication. Current detection methodologies for P. acnes involve prolonged anaerobic cultures that can take up to three weeks before findings can be reported. Our aim was to develop a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) approach that is both sensitive and specific to P. acnes that would enable a 24-hour turnaround between biopsy and results. Comparisons between the 16S ribosomal sequences of P. acnes and closely related bacteria identified two unique regions in P.acnes to which PCR primers were designed. Additionally, two unique restriction enzyme cut sites for HaeIII were identified within this amplicon. To test the PCR method, arthroscopic surgical biopsies were mechanically homogenised and boiled for 20 minutes to lyse the cellular membranes. PCR was performed using standard conditions followed by a one hour HaeIII enzymatic digest of the PCR product. Resultant fragments were visualised on polyacrylamide gels stained with ethidium bromide. All experiments included no-template controls to rule out reagent contamination and independently confirmed P. acnes DNA as a positive control. Serial dilutions of P. acnes cultures in Robertson's cooked-meat broth and spectrophotometric analysis of cellular concentration were used to assess the sensitivity of the PCR reaction. A unique 564 base-pair PCR amplicon was derived from different strains of P. acnes. This amplicon was confirmed as P. acnes DNA by gel excision and DNA sequencing. HaeIII digests of the amplicon yielded 3 restriction fragments at the sizes predicted by in silico analyses. Sensitivity testing confirmed that as few as 10 P. acnes cells in a 50µl reaction volume could be detected using this assay. P. acnes was also detected in surgical biopsy samples. P. acnes infections following shoulder arthroplasty are a serious complication placing a burden on the healthcare system and the patient due to the lengthy surgical revision process that follows. The infections are also difficult to diagnose. This unique assay combines the sensitivity of PCR with the specificity of RFLP mapping to specifically identify P. acnes in surgical isolates. We anticipate that this assay will allow us to determine if a biopsy is P. acnes positive within 24-hours of sampling, allowing for more aggressive antibiotic therapy and monitoring to avoid implant failure and revision surgery. Additionally, this PCR-RFLP method may decrease the false positive rate of extended length cultures due to P. acnes contamination

Cell-scaffold based cartilage tissue engineering strategies provide the potential to restore long-term function to damaged articular cartilage. A major hurdle in such strategies is the adequate (uniform and sufficient) population of porous 3D scaffolds with cells, but more importantly, the generation of engineered tissue of sufficient quality of clinically relevant size. We describe a novel approach to engineer cartilage grafts using pre-differentiated micro-mass cartilage pellets, integrated into specifically designed 3D plotted scaffolds. Expanded (P2) human nasal chondrocytes (HNCs) or bone marrow-derived mesenchymal stem cells (MSCs) from 3 donors (age 47–62 years) were micro-mass cell pellet cultivated at 5 × 105 cells/pellet for 4 days. Subsequently, pellets were integrated into degradable 3D Printed polymer (PEGT/PBT) scaffolds with 1mm fibre spacing. Constructs were cultured dynamically in spinner flasks for a total of 21 days. As a pellet-free control, expanded HNCs were spinner flask seeded into PEGT/PBT fibre plotted scaffolds. Constructs were assessed via histology (Safranin-O staining), biochemistry (glycosaminoglycan (GAG) and DNA content) and collagen type I and II mRNA expression. After 4 days, micro-mass cultured pellets could be successfully integrated into the fibre plotted scaffolds. DNA content of pellet integrated constructs was 4.0-fold±1.3 higher compared to single seeded constructs. At day 21, cartilage tissue was uniformly distributed throughout pellet integrated scaffolds, with minimal cell necrosis observed within the core. GAG/DNA and collagen type II mRNA expression were significantly higher (2.5-fold±0.5 and 3.1-fold±0.4 respectively) in pellet versus single cell seeded constructs. Furthermore, compared to single cell, the pellet seeded constructs contained significantly more total GAG and DNA (1.6-fold±0.1 and 3.1-fold±1.0 respectively). We developed a novel 3D tissue assembly approach for cartilage tissue engineering which significantly improved the seeding efficiency (∼100%), as well as tissue uniformity and integrity compared to more traditional seeding approaches using single cell suspensions. Furthermore, the integration of micro-mass cell pellets into 3D plotted PEGT/PBT scaffolds significantly improved the amount and quality of tissue engineered cartilage

Aim. We used a polymerase chain reaction (PCR) lateral flow assay1) to rapidly diagnose joint infection. We evaluated the usefulness of multiplex-PCR (PCR lateral flow assay: PCR-LF) using detailed clinical data. Method. A total of 35 synovial fluid samples were collected from 26 patients in whom bacterial infection was suspected, including 22 from knee joints, 11 from hip joints, and 2 from other joints. After purifying the DNA from the samples, multiplex PCR targeting two MRSA-associated genes (femA and mecA) and the bacterial 16S rRNA gene was performed. Amplified gene fragments were specifically detected with DNA probes immobilized on stick devices through DNA-DNA hybridization and visualization, enabling diagnosis of MRSA, MSSA, MRCNS, gram-positive, and/or gram-negative bacterial infection. Genetic identification of bacteria by determining the 16S rRNA gene sequence was also performed using multiplex PCR-positive samples. Finally, the usefulness of our PCR-LF method was evaluated using detailed clinical data. Results. The results of PCR-LF were 9 gram-positive and 1 gram-negative bacterial infections. Eleven bacterial species were identified based on 16S rRNA gene sequences. Ten (90.9%)of the eleven samples (bacterial species) were identified using our PCR-LF. Five samples were detected in bacterial cultures; two are MSSA, one is Streptococcus agalactiae, one is Escherichia coli, one is Prevotella oralis. We diagnosed 6 samples as clinical infections. Therefore, the sensitivity and specificity of the culture tests were 83% and 100%, respectively, while for PCR-LF, these values were 83% and 83%. Conclusions. PCR-LF is highly sensitive and effective for the rapid diagnosis of joint infection; however, dead bacteria may also be detected. Moreover, because the target bacterial species are limited, clinical diagnosis based on the results of multiple examinations is necessary