The Experts below are selected from a list of 2169 Experts worldwide ranked by ideXlab platform
Bingbing Jiang - One of the best experts on this subject based on the ideXlab platform.
-
polypeptide nanocoatings for preventing dental and Orthopaedic Device associated infection ph induced antibiotic capture release and antibiotic efficacy
Journal of Biomedical Materials Research Part B, 2009Co-Authors: Bingbing JiangAbstract:Implant-associated infection is one of the most common and problematic complications for dental and Orthopaedic patients. Modification of currently used implant surfaces aimed at bestowing them with antibacterial properties is a promising approach in the development of new biomaterials. In this study, a novel nanotechnology, that is, electrostatic self-assembly, was developed to construct biomimetic polypeptide nanocoatings on commonly used metal implants. A model antibacterial drug, cefazolin, was captured in the polypeptide nanocoating and its release was studied. We have shown that the capture and release of cefazolin was pH-induced and could be controlled, and the developed antibiotic-incorporated polypeptide multilayer nanocoatings could prevent Staphylococcus aureus colonization thus showing great potential for preventing implant-associated infection. © 2007 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2009
-
polypeptide nanocoatings for preventing dental and Orthopaedic Device associated infection ph induced antibiotic capture release and antibiotic efficacy
Journal of Biomedical Materials Research Part B, 2009Co-Authors: Bingbing JiangAbstract:Implant-associated infection is one of the most common and problematic complications for dental and Orthopaedic patients. Modification of currently used implant surfaces aimed at bestowing them with antibacterial properties is a promising approach in the development of new biomaterials. In this study, a novel nanotechnology, that is, electrostatic self-assembly, was developed to construct biomimetic polypeptide nanocoatings on commonly used metal implants. A model antibacterial drug, cefazolin, was captured in the polypeptide nanocoating and its release was studied. We have shown that the capture and release of cefazolin was pH-induced and could be controlled, and the developed antibiotic-incorporated polypeptide multilayer nanocoatings could prevent Staphylococcus aureus colonization thus showing great potential for preventing implant-associated infection.
Andrew Brent - One of the best experts on this subject based on the ideXlab platform.
-
tissue culture and sonication for diagnosis of prosthetic joint and Orthopaedic Device related infection
Orthopaedic Proceedings, 2018Co-Authors: Andrew Brent, M A Mcnally, Lucinda Barrett, Maria Dudareva, Mel Figtree, R Colledge, Robert Newnham, Philip Bejon, Aimee R Taylor, B AtkinsAbstract:Collection of 4–5 independent peri-prosthetic tissue samples is recommended for microbiological diagnosis of prosthetic joint infections. Sonication of explanted prostheses has also been shown to increase microbiological yield in some centres. We compared sonication with standard tissue sampling for diagnosis of prosthetic joint and other Orthopaedic Device related infections.We used standard protocols for sample collection, tissue culture and sonication. Positive tissue culture was defined as isolation of a phenotypically indistinguishable organism from ≥2 samples; and positive sonication culture as isolation of an organism at ≥50 cfu/ml. We compared the diagnostic performance of each method against an established clinical definition of infection (Trampuz 2011), and against a composite clinical and microbiological definition of infection based on international consensus (Gehrke & Parvizi 2013).350 specimens were received for sonication, including joint prostheses (160), exchangeable components (76), othe...
-
metagenomic sequencing for Orthopaedic Device related infection
Orthopaedic Proceedings, 2018Co-Authors: Teresa L Street, Nicholas D Sanderson, Bridget L Atkins, Andrew Brent, Kevin Cole, Dona Foster, M A Mcnally, Sarah Oakley, Leon Peto, A TaylorAbstract: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 obtai...
-
metagenomic sequencing for Orthopaedic Device related infection
Journal of Bone and Joint Surgery-british Volume, 2017Co-Authors: Teresa L Street, Nicholas D Sanderson, Bridget L Atkins, Andrew Brent, Kevin Cole, Dona Foster, M A Mcnally, Sarah Oakley, Leon Peto, A TaylorAbstract: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.
-
real time analysis of nanopore based metagenomic sequencing from Orthopaedic Device infection
bioRxiv, 2017Co-Authors: Nicholas D Sanderson, Bridget L Atkins, Andrew Brent, Dona Foster, M A Mcnally, Sarah Oakley, A Taylor, Teresa L Street, Jeremy Swann, T PetoAbstract:Prosthetic joint infections are clinically difficult to diagnose and treat. Previously, we demonstrated metagenomic sequencing on an Illumina MiSeq replicates the findings of current gold standard microbiological diagnostic techniques. Nanopore sequencing offers advantages in speed of detection over MiSeq. Here, we compare direct-from-clinical-sample metagenomic Illumina sequencing with Nanopore sequencing, and report a real-time analytical pathway for Nanopore sequence data, designed for detecting bacterial composition of prosthetic joint infections. DNA was extracted from the sonication fluids of seven explanted Orthopaedic Devices, and additionally from two culture negative controls, and was sequenced on the Oxford Nanopore Technologies MinION platform. A specific analysis pipeline was assembled to overcome the challenges of identifying the true infecting pathogen, given high levels of host contamination and unavoidable background lab and kit contamination. The majority of DNA classified (>90%) was host contamination and discarded. Using negative control filtering thresholds, the species identified corresponded with both routine microbiological diagnosis and MiSeq results. By analysing sequences in real time, causes of infection were robustly detected within minutes from initiation of sequencing. We demonstrate initial proof of concept that metagenomic MinION sequencing can provide rapid, accurate diagnosis for prosthetic joint infections. We demonstrate a novel, scalable pipeline for real-time analysis of MinION sequence data. The high proportion of human DNA in extracts prevents full genome analysis from complete coverage, and methods to reduce this could increase genome depth and allow antimicrobial resistance profiling.
-
molecular diagnosis of Orthopaedic Device infection direct from sonication fluid by metagenomic sequencing
bioRxiv, 2017Co-Authors: Teresa Street, Nicholas D Sanderson, Bridget L Atkins, Andrew Brent, Kevin Cole, Dona Foster, M A Mcnally, Sarah Oakley, Leon Peto, A TaylorAbstract: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. However, current techniques can have relatively low sensitivity, with prior antimicrobial therapy and infection by fastidious organisms influencing results. We assessed if metagenomic sequencing of complete bacterial DNA extracts obtained direct from sonication fluid can provide an alternative rapid and sensitive tool for diagnosis of PJI. We compared metagenomic sequencing with standard aerobic and anaerobic culture in 97 sonication fluid samples from prosthetic joint and other Orthopaedic Device infections. Reads from Illumina MiSeq sequencing were taxonomically classified using Kraken. Using 50 samples (derivation set), we determined optimal thresholds for the number and proportion of bacterial reads required to identify an infection and validated our findings in 47 independent 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 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. Rigorous laboratory good practice was required to prevent bacterial DNA contamination. We demonstrate metagenomic sequencing can provide accurate diagnostic information in PJI. Our findings combined with increasing availability of portable, random-access sequencing technology offers the potential to translate metagenomic sequencing into a rapid diagnostic tool in PJI.
M A Mcnally - One of the best experts on this subject based on the ideXlab platform.
-
tissue culture and sonication for diagnosis of prosthetic joint and Orthopaedic Device related infection
Orthopaedic Proceedings, 2018Co-Authors: Andrew Brent, M A Mcnally, Lucinda Barrett, Maria Dudareva, Mel Figtree, R Colledge, Robert Newnham, Philip Bejon, Aimee R Taylor, B AtkinsAbstract:Collection of 4–5 independent peri-prosthetic tissue samples is recommended for microbiological diagnosis of prosthetic joint infections. Sonication of explanted prostheses has also been shown to increase microbiological yield in some centres. We compared sonication with standard tissue sampling for diagnosis of prosthetic joint and other Orthopaedic Device related infections.We used standard protocols for sample collection, tissue culture and sonication. Positive tissue culture was defined as isolation of a phenotypically indistinguishable organism from ≥2 samples; and positive sonication culture as isolation of an organism at ≥50 cfu/ml. We compared the diagnostic performance of each method against an established clinical definition of infection (Trampuz 2011), and against a composite clinical and microbiological definition of infection based on international consensus (Gehrke & Parvizi 2013).350 specimens were received for sonication, including joint prostheses (160), exchangeable components (76), othe...
-
metagenomic sequencing for Orthopaedic Device related infection
Orthopaedic Proceedings, 2018Co-Authors: Teresa L Street, Nicholas D Sanderson, Bridget L Atkins, Andrew Brent, Kevin Cole, Dona Foster, M A Mcnally, Sarah Oakley, Leon Peto, A TaylorAbstract: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 obtai...
-
metagenomic sequencing for Orthopaedic Device related infection
Journal of Bone and Joint Surgery-british Volume, 2017Co-Authors: Teresa L Street, Nicholas D Sanderson, Bridget L Atkins, Andrew Brent, Kevin Cole, Dona Foster, M A Mcnally, Sarah Oakley, Leon Peto, A TaylorAbstract: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.
-
real time analysis of nanopore based metagenomic sequencing from Orthopaedic Device infection
bioRxiv, 2017Co-Authors: Nicholas D Sanderson, Bridget L Atkins, Andrew Brent, Dona Foster, M A Mcnally, Sarah Oakley, A Taylor, Teresa L Street, Jeremy Swann, T PetoAbstract:Prosthetic joint infections are clinically difficult to diagnose and treat. Previously, we demonstrated metagenomic sequencing on an Illumina MiSeq replicates the findings of current gold standard microbiological diagnostic techniques. Nanopore sequencing offers advantages in speed of detection over MiSeq. Here, we compare direct-from-clinical-sample metagenomic Illumina sequencing with Nanopore sequencing, and report a real-time analytical pathway for Nanopore sequence data, designed for detecting bacterial composition of prosthetic joint infections. DNA was extracted from the sonication fluids of seven explanted Orthopaedic Devices, and additionally from two culture negative controls, and was sequenced on the Oxford Nanopore Technologies MinION platform. A specific analysis pipeline was assembled to overcome the challenges of identifying the true infecting pathogen, given high levels of host contamination and unavoidable background lab and kit contamination. The majority of DNA classified (>90%) was host contamination and discarded. Using negative control filtering thresholds, the species identified corresponded with both routine microbiological diagnosis and MiSeq results. By analysing sequences in real time, causes of infection were robustly detected within minutes from initiation of sequencing. We demonstrate initial proof of concept that metagenomic MinION sequencing can provide rapid, accurate diagnosis for prosthetic joint infections. We demonstrate a novel, scalable pipeline for real-time analysis of MinION sequence data. The high proportion of human DNA in extracts prevents full genome analysis from complete coverage, and methods to reduce this could increase genome depth and allow antimicrobial resistance profiling.
-
molecular diagnosis of Orthopaedic Device infection direct from sonication fluid by metagenomic sequencing
bioRxiv, 2017Co-Authors: Teresa Street, Nicholas D Sanderson, Bridget L Atkins, Andrew Brent, Kevin Cole, Dona Foster, M A Mcnally, Sarah Oakley, Leon Peto, A TaylorAbstract: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. However, current techniques can have relatively low sensitivity, with prior antimicrobial therapy and infection by fastidious organisms influencing results. We assessed if metagenomic sequencing of complete bacterial DNA extracts obtained direct from sonication fluid can provide an alternative rapid and sensitive tool for diagnosis of PJI. We compared metagenomic sequencing with standard aerobic and anaerobic culture in 97 sonication fluid samples from prosthetic joint and other Orthopaedic Device infections. Reads from Illumina MiSeq sequencing were taxonomically classified using Kraken. Using 50 samples (derivation set), we determined optimal thresholds for the number and proportion of bacterial reads required to identify an infection and validated our findings in 47 independent 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 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. Rigorous laboratory good practice was required to prevent bacterial DNA contamination. We demonstrate metagenomic sequencing can provide accurate diagnostic information in PJI. Our findings combined with increasing availability of portable, random-access sequencing technology offers the potential to translate metagenomic sequencing into a rapid diagnostic tool in PJI.
R G Richards - One of the best experts on this subject based on the ideXlab platform.
-
A large animal model for a failed two-stage revision of intramedullary nail-related infection by methicillin-resistant Staphylococcus aureus
AO Research Institute Davos, 2017Co-Authors: T F Moriarty, T Schmid, V Post, E Samara, S Kates, Em Schwarz, S Zeiter, R G RichardsAbstract:The treatment of chronic Orthopaedic Device-associated infection (ODRI) often requires multiple surgeries and prolonged antibiotic therapy. Despite this extensive treatment protocol, the procedure is associated with significant failure rates. Currently, no large animal model is available that recapitulates a failed revision. Therefore, our aim was to establish a large animal model for failed treatment of an ODRI in order to serve as a testbed for future interventional strategies. Adult Swiss Alpine sheep received an intramedullary nail in the tibia and a localised inoculum of either a methicillin-sensitive or methicillin-resistant Staphylococcus aureus (MSSA, MRSA respectively). After 8 weeks, when chronic infection had been established, the animals underwent a staged revision with debridement and temporary placement of an antibiotic-loaded cement spacer. Antibiotics were delivered systemically in a standard or pathogen-adapted manner. Debridement and implant exchange alone failed to treat the MSSA infection. Neither local therapy alone nor systemic therapy alone were effective in resolving infection with MSSA, but a combination of local and systemic therapy was effective against it. MRSA infection was not resolved by the combination of local and systemic antibiotics (standard or pathogen-adapted). A model for failed revision of MRSA infection is described despite the use of local and systemic antibiotics. Novel interventions may be assessed using this model, including antibiotic and non-antibiotic interventions
-
adhesion formation of primary human osteoblasts and the functional response of mesenchymal stem cells to 330 nm deep microgrooves
Journal of the Royal Society Interface, 2008Co-Authors: Manus J P Biggs, R G Richards, Richard O C Oreffo, S Mcfarlane, C D W Wilkinson, Matthew J DalbyAbstract:The surface microtexture of an Orthopaedic Device can regulate cellular adhesion, a process fundamental in the initiation of osteoinduction and osteogenesis. Advances in fabrication techniques have evolved to include the field of surface modification; in particular, nanotechnology has allowed for the development of experimental nanoscale substrates for investigation into cell nanofeature interactions. Here primary human osteoblasts (HOBs) were cultured on ordered nanoscale groove/ridge arrays fabricated by photolithography. Grooves were 330 nm deep and either 10, 25 or 100 μm in width. Adhesion subtypes in HOBs were quantified by immunofluorescent microscopy and cell–substrate interactions were investigated via immunocytochemistry with scanning electron microscopy. To further investigate the effects of these substrates on cellular function, 1.7 K gene microarray analysis was used to establish gene regulation profiles of mesenchymal stem cells cultured on these nanotopographies. Nanotopographies significantly affected the formation of focal complexes (FXs), focal adhesions (FAs) and supermature adhesions (SMAs). Planar control substrates induced widespread adhesion formation; 100 μm wide groove/ridge arrays did not significantly affect adhesion formation yet induced upregulation of genes involved in skeletal development and increased osteospecific function; 25 μm wide groove/ridge arrays were associated with a reduction in SMA and an increase in FX formation; and 10 μm wide groove/ridge arrays significantly reduced osteoblast adhesion and induced an interplay of up- and downregulation of gene expression. This study indicates that groove/ridge topographies are important modulators of both cellular adhesion and osteospecific function and, critically, that groove/ridge width is important in determining cellular response.
-
an introduction to staphylococcus aureus and techniques for identifying and quantifying s aureus adhesins in relation to adhesion to biomaterials review
European Cells & Materials, 2002Co-Authors: Llinos G Harris, S J Foster, R G RichardsAbstract:The ability of Staphylococcus aureus to adhere to the extracellular matrix and plasma proteins deposited on biomaterials is a significant factor in the pathogenesis of Orthopaedic-Device related infections. S. aureus possesses many adhesion proteins on its surface, but it is not known how they interact with each other to form stable interactions with the substrate. A novel method was developed for extracting adhesins from the S. aureus cell wall, which could then be further analysed. The protocol involves using a FastPrep instrument to mechanically disrupt the cell walls resulting in native cell walls. Ionically and covalently bound proteins were then solubilised using sodium dodecyl sulphate (SDS) and lysostaphin, respectively. Western blot analysis of covalently bound proteins using anti-protein A and anti-clumping factor A sera showed that S. aureus produces most surface proteins in early growth, and less in post-exponential and stationary growth. Immuno-gold labelling of protein A, and clumping factor A was observed all over the bacteria and showed no distinct surface distribution pattern. However, this labelling showed expression of surface associated proteins varied in a growth-phase dependent and cell-density dependent manner.
A Taylor - One of the best experts on this subject based on the ideXlab platform.
-
metagenomic sequencing for Orthopaedic Device related infection
Orthopaedic Proceedings, 2018Co-Authors: Teresa L Street, Nicholas D Sanderson, Bridget L Atkins, Andrew Brent, Kevin Cole, Dona Foster, M A Mcnally, Sarah Oakley, Leon Peto, A TaylorAbstract: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 obtai...
-
metagenomic sequencing for Orthopaedic Device related infection
Journal of Bone and Joint Surgery-british Volume, 2017Co-Authors: Teresa L Street, Nicholas D Sanderson, Bridget L Atkins, Andrew Brent, Kevin Cole, Dona Foster, M A Mcnally, Sarah Oakley, Leon Peto, A TaylorAbstract: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.
-
real time analysis of nanopore based metagenomic sequencing from Orthopaedic Device infection
bioRxiv, 2017Co-Authors: Nicholas D Sanderson, Bridget L Atkins, Andrew Brent, Dona Foster, M A Mcnally, Sarah Oakley, A Taylor, Teresa L Street, Jeremy Swann, T PetoAbstract:Prosthetic joint infections are clinically difficult to diagnose and treat. Previously, we demonstrated metagenomic sequencing on an Illumina MiSeq replicates the findings of current gold standard microbiological diagnostic techniques. Nanopore sequencing offers advantages in speed of detection over MiSeq. Here, we compare direct-from-clinical-sample metagenomic Illumina sequencing with Nanopore sequencing, and report a real-time analytical pathway for Nanopore sequence data, designed for detecting bacterial composition of prosthetic joint infections. DNA was extracted from the sonication fluids of seven explanted Orthopaedic Devices, and additionally from two culture negative controls, and was sequenced on the Oxford Nanopore Technologies MinION platform. A specific analysis pipeline was assembled to overcome the challenges of identifying the true infecting pathogen, given high levels of host contamination and unavoidable background lab and kit contamination. The majority of DNA classified (>90%) was host contamination and discarded. Using negative control filtering thresholds, the species identified corresponded with both routine microbiological diagnosis and MiSeq results. By analysing sequences in real time, causes of infection were robustly detected within minutes from initiation of sequencing. We demonstrate initial proof of concept that metagenomic MinION sequencing can provide rapid, accurate diagnosis for prosthetic joint infections. We demonstrate a novel, scalable pipeline for real-time analysis of MinION sequence data. The high proportion of human DNA in extracts prevents full genome analysis from complete coverage, and methods to reduce this could increase genome depth and allow antimicrobial resistance profiling.
-
molecular diagnosis of Orthopaedic Device infection direct from sonication fluid by metagenomic sequencing
bioRxiv, 2017Co-Authors: Teresa Street, Nicholas D Sanderson, Bridget L Atkins, Andrew Brent, Kevin Cole, Dona Foster, M A Mcnally, Sarah Oakley, Leon Peto, A TaylorAbstract: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. However, current techniques can have relatively low sensitivity, with prior antimicrobial therapy and infection by fastidious organisms influencing results. We assessed if metagenomic sequencing of complete bacterial DNA extracts obtained direct from sonication fluid can provide an alternative rapid and sensitive tool for diagnosis of PJI. We compared metagenomic sequencing with standard aerobic and anaerobic culture in 97 sonication fluid samples from prosthetic joint and other Orthopaedic Device infections. Reads from Illumina MiSeq sequencing were taxonomically classified using Kraken. Using 50 samples (derivation set), we determined optimal thresholds for the number and proportion of bacterial reads required to identify an infection and validated our findings in 47 independent 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 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. Rigorous laboratory good practice was required to prevent bacterial DNA contamination. We demonstrate metagenomic sequencing can provide accurate diagnostic information in PJI. Our findings combined with increasing availability of portable, random-access sequencing technology offers the potential to translate metagenomic sequencing into a rapid diagnostic tool in PJI.
