scholarly journals Molecular diagnosis of orthopaedic device infection direct from sonication fluid by metagenomic sequencing

2017 ◽  
Author(s):  
Teresa L. Street ◽  
Nicholas D. Sanderson ◽  
Bridget L. Atkins ◽  
Andrew J. Brent ◽  
Kevin Cole ◽  
...  
Keyword(s):  
Diagnostic Information ◽  
Species Level ◽  
Metagenomic Sequencing ◽  
Tissue Samples ◽  
Bacterial Dna ◽  
Dna Contamination ◽  
Prosthetic Joint ◽  
Device Infection ◽  
Fluid Culture ◽  
Orthopaedic Device

AbstractCulture 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.

scholarly journals Molecular Diagnosis of Orthopedic-Device-Related Infection Directly from Sonication Fluid by Metagenomic Sequencing

2017 ◽  
Vol 55 (8) ◽  
pp. 2334-2347 ◽  
Author(s):  
Teresa L. Street ◽  
Nicholas D. Sanderson ◽  
Bridget L. Atkins ◽  
Andrew J. Brent ◽  
Kevin Cole ◽  
...  
Keyword(s):  
Good Practice ◽  
Illumina Miseq ◽  
Diagnostic Information ◽  
Species Level ◽  
Metagenomic Sequencing ◽  
Tissue Samples ◽  
Orthopedic Device ◽  
Dna Contamination ◽  
Prosthetic Joint ◽  
Prosthetic Joint Infections

ABSTRACTCulture 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 culture of sonication fluid from 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 total 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 orthopedic device infections. Reads from Illumina MiSeq sequencing 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. Compared to results from sonication fluid culture, the species-level sensitivity of metagenomic sequencing was 61/69 (88%; 95% confidence interval [CI], 77 to 94%; for derivation samples 35/38 [92%; 95% CI, 79 to 98%]; for validation samples, 26/31 [84%; 95% CI, 66 to 95%]), and genus-level sensitivity was 64/69 (93%; 95% CI, 84 to 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%; 95% CI, 79 to 93%; for derivation samples, 43/50 [86%; 95% CI, 73 to 94%]; for validation samples, 42/47 [89%; 95% CI, 77 to 96%]). High levels of human DNA contamination were seen despite the use of laboratory methods to remove it. Rigorous laboratory good practice was required to minimize bacterial DNA contamination. We demonstrate that metagenomic sequencing can provide accurate diagnostic information in PJI. Our findings, combined with the increasing availability of portable, random-access sequencing technology, offer the potential to translate metagenomic sequencing into a rapid diagnostic tool in PJI.

scholarly journals Evaluation of the CosmosID Bioinformatics Platform for Prosthetic Joint-Associated Sonicate Fluid Shotgun Metagenomic Data Analysis

2018 ◽  
Vol 57 (2) ◽  
Author(s):  
Qun Yan ◽  
Yu Mi Wi ◽  
Matthew J. Thoendel ◽  
Yash S. Raval ◽  
Kerryl E. Greenwood-Quaintance ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Metagenomic Data ◽  
Metagenomic Sequencing ◽  
Antibacterial Resistance ◽  
Sequencing Data ◽  
Bacterial Detection ◽  
Shotgun Metagenomic Sequencing ◽  
Prosthetic Joint ◽  
Validation Set ◽  
Fluid Culture

ABSTRACT We previously demonstrated that shotgun metagenomic sequencing can detect bacteria in sonicate fluid, providing a diagnosis of prosthetic joint infection (PJI). A limitation of the approach that we used is that data analysis was time-consuming and specialized bioinformatics expertise was required, both of which are barriers to routine clinical use. Fortunately, automated commercial analytic platforms that can interpret shotgun metagenomic data are emerging. In this study, we evaluated the CosmosID bioinformatics platform using shotgun metagenomic sequencing data derived from 408 sonicate fluid samples from our prior study with the goal of evaluating the platform vis-à-vis bacterial detection and antibiotic resistance gene detection for predicting staphylococcal antibacterial susceptibility. Samples were divided into a derivation set and a validation set, each consisting of 204 samples; results from the derivation set were used to establish cutoffs, which were then tested in the validation set for identifying pathogens and predicting staphylococcal antibacterial resistance. Metagenomic analysis detected bacteria in 94.8% (109/115) of sonicate fluid culture-positive PJIs and 37.8% (37/98) of sonicate fluid culture-negative PJIs. Metagenomic analysis showed sensitivities ranging from 65.7 to 85.0% for predicting staphylococcal antibacterial resistance. In conclusion, the CosmosID platform has the potential to provide fast, reliable bacterial detection and identification from metagenomic shotgun sequencing data derived from sonicate fluid for the diagnosis of PJI. Strategies for metagenomic detection of antibiotic resistance genes for predicting staphylococcal antibacterial resistance need further development.

scholarly journals Optimized DNA isolation method for microbiome analysis of human tissues

2020 ◽  
Author(s):  
Carlijn Bruggeling ◽  
Daniel R. Garza ◽  
Soumia Achouiti ◽  
Wouter Mes ◽  
Bas E. Dutilh ◽  
...  
Keyword(s):  
Relative Abundance ◽  
Dna Isolation ◽  
Diversity Index ◽  
Wide Spectrum ◽  
Host Cells ◽  
Metagenomic Sequencing ◽  
Tissue Samples ◽  
Unifrac Distance ◽  
Bacterial Dna ◽  
Dnase Treatment

AbstractRecent advances in microbiome sequencing have rendered new insights into the role of the microbiome in human health with potential clinical implications. Unfortunately, developments in the field of tissue microbiomes have been hampered by the presence of host DNA in isolates which interferes with the analysis of the bacterial content. Here, we present a DNA isolation protocol from tissue samples including reduction of host DNA without distortion of microbial abundance profiles. We evaluated which concentrations of Triton and saponin lyse host cells and leave bacterial cells intact, which was combined with DNAse treatment to deplete released host DNA. We applied our protocol to extract microbial DNA from ex vivo and in vivo acquired human colon biopsies (∼2-5 mm in size) and assessed the relative abundance of bacterial and human DNA by qPCR. Saponin at a concentration of 0.0125% in PBS lysed host cells, resulting in a 4.5-fold enrichment of bacterial DNA while preserving the relative abundance of Firmicutes, Bacteroidetes, γ-Proteobacteria and Actinobacteria. Our protocol combined with shotgun metagenomic sequencing revealed a colon tissue microbiome profile with a Shannon diversity index of 3.2 and an UniFrac distance of 0.54, which is comparable to reported numbers based on amplicon sequencing. Hereby, we present the first protocol for enriching bacterial DNA from tissue biopsies that allows efficient isolation of rigid Gram-positive bacteria without depleting the more sensitive Gram-negative bacteria. Our protocol facilitates analysis of a wide spectrum of bacteria of clinical tissue samples improving their applicability for microbiome research.

scholarly journals Direct Detection and Identification of Prosthetic Joint Infection Pathogens in Synovial Fluid by Metagenomic Shotgun Sequencing

2018 ◽  
Vol 56 (9) ◽  
Author(s):  
Morgan I. Ivy ◽  
Matthew J. Thoendel ◽  
Patricio R. Jeraldo ◽  
Kerryl E. Greenwood-Quaintance ◽  
Arlen D. Hanssen ◽  
...  
Keyword(s):  
Synovial Fluid ◽  
Prosthetic Joint Infection ◽  
Joint Infection ◽  
Shotgun Sequencing ◽  
Species Level ◽  
Prosthetic Joint ◽  
Synovial Fluid Culture ◽  
Culture Negative ◽  
Aseptic Failure ◽  
Fluid Culture

ABSTRACTMetagenomic shotgun sequencing has the potential to transform how serious infections are diagnosed by offering universal, culture-free pathogen detection. This may be especially advantageous for microbial diagnosis of prosthetic joint infection (PJI) by synovial fluid analysis since synovial fluid cultures are not universally positive and since synovial fluid is easily obtained preoperatively. We applied a metagenomics-based approach to synovial fluid in an attempt to detect microorganisms in 168 failed total knee arthroplasties. Genus- and species-level analyses of metagenomic sequencing yielded the known pathogen in 74 (90%) and 68 (83%) of the 82 culture-positive PJIs analyzed, respectively, with testing of two (2%) and three (4%) samples, respectively, yielding additional pathogens not detected by culture. For the 25 culture-negative PJIs tested, genus- and species-level analyses yielded 19 (76%) and 21 (84%) samples with insignificant findings, respectively, and 6 (24%) and 4 (16%) with potential pathogens detected, respectively. Genus- and species-level analyses of the 60 culture-negative aseptic failure cases yielded 53 (88%) and 56 (93%) cases with insignificant findings and 7 (12%) and 4 (7%) with potential clinically significant organisms detected, respectively. There was one case of aseptic failure with synovial fluid culture growth; metagenomic analysis showed insignificant findings, suggesting possible synovial fluid culture contamination. Metagenomic shotgun sequencing can detect pathogens involved in PJI when applied to synovial fluid and may be particularly useful for culture-negative cases.

scholarly journals Real-time analysis of nanopore-based metagenomic sequencing from orthopaedic device infection

2017 ◽  
Author(s):  
Nicholas D Sanderson ◽  
Teresa L Street ◽  
Dona Foster ◽  
Jeremy Swann ◽  
Bridget L. Atkins ◽  
...  
Keyword(s):  
Real Time ◽  
Sequence Data ◽  
Metagenomic Sequencing ◽  
Nanopore Sequencing ◽  
Time Analysis ◽  
Real Time Analysis ◽  
Joint Infections ◽  
Prosthetic Joint ◽  
Device Infection ◽  
Prosthetic Joint Infections

AbstractProsthetic 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.

scholarly journals Clinical Metagenomic Sequencing for Species Identification and Antimicrobial Resistance Prediction in Orthopaedic Device Infection

2021 ◽  
Author(s):  
Teresa L Street ◽  
Nicholas D Sanderson ◽  
Camille Kolenda ◽  
James Kavanagh ◽  
Hayleah Pickford ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Sequence Data ◽  
Revision Arthroplasty ◽  
Metagenomic Data ◽  
Metagenomic Sequencing ◽  
Human Dna ◽  
Oxford Nanopore ◽  
Device Infection ◽  
Infection Types ◽  
Orthopaedic Device

Background Diagnosis of orthopaedic device-related infection is challenging, and causative pathogens may be difficult to culture. Metagenomic sequencing can diagnose infections without culture, but attempts to detect antimicrobial resistance (AMR) determinants using metagenomic data have been less successful. Human DNA depletion may maximise the amount of microbial DNA sequence data available for analysis. Methods Human DNA depletion by saponin was tested in 115 sonication fluid samples generated following revision arthroplasty surgery, comprising 67 where pathogens were detected by culture and 48 culture-negative samples. Metagenomic sequencing was performed on the Oxford Nanopore Technologies GridION platform. Filtering thresholds for detection of true species versus contamination or taxonomic misclassification were determined. Mobile and chromosomal genetic AMR determinants were identified in Staphylococcus aureus-positive samples. Results Of 114 samples generating sequence data, species-level sensitivity of metagenomic sequencing was 49/65 (75%; 95%CI 63-85%) and specificity 103/114 (90%; 95%CI 83-95%) compared with culture. Saponin treatment reduced the proportion of human bases sequenced in comparison to 5um filtration from a median (IQR) 98.1% (87.0%-99.9%) to 11.9% (0.4%-67.0%), improving reference genome coverage at 10-fold depth from 18.7% (0.30%-85.7%) to 84.3% (12.9%-93.8%). Metagenomic sequencing predicted 13/15 (87%) resistant and 74/74 (100%) susceptible phenotypes where sufficient data were available for analysis. Conclusions Metagenomic nanopore sequencing coupled with human DNA depletion has the potential to detect AMR in addition to species detection in orthopaedic device-related infection. Further work is required to develop pathogen-agnostic human DNA depletion methods, improving AMR determinant detection and allowing its application to other infection types.

scholarly journals The Effect of Genomic DNA Contamination on the Detection of Circulating Long Non-Coding RNAs: The Paradigm of MALAT1

Diagnostics ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 1160
Author(s):  
Athina N. Markou ◽  
Stavroula Smilkou ◽  
Emilia Tsaroucha ◽  
Evi Lianidou
Keyword(s):  
False Positive ◽  
Tissue Samples ◽  
Dna Contamination ◽  
Dnase Treatment ◽  
Circular Dnas ◽  
Non Coding Rnas ◽  
Nsclc Patients ◽  
Treatment Step ◽  
Primary Tissue ◽  
Positive Results

The presence of contaminating gDNA in RNA preparations is a frequent cause of false positives in RT-PCR-based analysis. However, in some cases, this cannot be avoided, especially when there are no exons–intron junctions in the lncRNA sequences. Due to the lack of exons in few of long noncoding RNAs (lncRNAs) and the lack of DNAse treatment step in most studies reported so far, serious questions are raised about the specificity of lncRNA detection and the potential of reporting false-positive results. We hypothesized that minute amounts of gDNA usually co-extracted with RNA could give false-positive signals since primers would specifically bind to gDNA due to the lack of junction. In the current study, we evaluated the effect of gDNA and other forms of DNA like extrachromosomal circular DNAs (eccDNAs) contamination and the importance of including a DNAse treatment step on lncRNAsexpression.As a model, we have chosen as one of the most widely studied lncRNAs in cancer namely MALAT1, which lacks exons. When we tested this hypothesis in plasma and primary tissue samples from NSCLC patients, our findings clearly indicated that results on MALAT1 expression are highly affected by the presence of DNA contamination and that the DNAse treatment step is absolutely necessary to avoid false positive results.

scholarly journals High Accuracy of Second Generation Unyvero ITI Automated Multiplex PCR Analysis of Joint Fluid Aspirate in Low-Grade Prosthetic Hip and Knee Joint Infections

2020 ◽  
Vol 8 (5_suppl4) ◽  
pp. 2325967120S0030
Author(s):  
Christian Suren ◽  
Susanne Feihl ◽  
Sabrina Cabric ◽  
Ingo Banke ◽  
Bernhard Haller ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Revision Surgery ◽  
Second Generation ◽  
Joint Fluid ◽  
Low Grade ◽  
Tissue Biopsy ◽  
Conventional Culture ◽  
Joint Infections ◽  
Prosthetic Joint ◽  
Fluid Culture

Aims and Objectives: A major obstacle for the treatment of prosthetic joint infections (PJIs) is the identification of the organism causing it. While the diagnostic criteria ruling PJIs in or out have become ever more accurate, detecting the causative pathogen(s) still relies mostly on conventional culture. The aim of this study was to evaluate the diagnostic potential of a second generation multiplex PCR assay (Unyvero ITI G2, Curetis AG, Holzgerlingen, Germany) used on synovial fluid specimens. Materials and Methods: Prospectively collected frozen joint fluid specimens from 26 patients undergoing arthroplasty revision surgery of the hip or knee for any reason were tested as per the manufacturer’s protocol. The sensitivity and specificity were calculated using the Chi-squared-test and a combination of the serum CRP level, leukocyte count, erythrocyte sedimentation rate, joint fluid culture, tissue biopsy culture, and tissue biopsy histology served as gold standard. Results: Of the 26 cases included in the study, 15 were infected and 11 aseptic. Conventional joint fluid culture showed a sensitivity 0.67 and a specificity of 0.91. Joint fluid multiplex PCR yielded a sensitivity of 0.8 and a specificity of 1.0. Conclusion: This is the first study to assess the second generation Unyvero ITI cartridge on joint fluid aspirate for the detection of prosthetic joint infection. In our setting, the method achieved a higher diagnostic accuracy than conventional culture. We conclude that, to improve pathogen detection before revision surgery, this method represents a valuable tool.

scholarly journals Accurate Reconstruction of Microbial Strains from Metagenomic Sequencing Using Representative Reference Genomes

2017 ◽  
Author(s):  
Zhemin Zhou ◽  
Nina Luhmann ◽  
Nabil-Fareed Alikhan ◽  
Christopher Quince ◽  
Mark Achtman
Keyword(s):  
Evaluation Studies ◽  
Species Level ◽  
Metagenomic Sequencing ◽  
Sequencing Data ◽  
Reference Databases ◽  
Microbial Strains ◽  
Taxonomic Assignments ◽  
Taxonomic Groups ◽  
Reference Genomes ◽  
Recent Evaluation

AbstractExploring the genetic diversity of microbes within the environment through metagenomic sequencing first requires classifying these reads into taxonomic groups. Current methods compare these sequencing data with existing biased and limited reference databases. Several recent evaluation studies demonstrate that current methods either lack sufficient sensitivity for species-level assignments or suffer from false positives, overestimating the number of species in the metagenome. Both are especially problematic for the identification of low-abundance microbial species, e. g. detecting pathogens in ancient metagenomic samples. We present a new method, SPARSE, which improves taxonomic assignments of metagenomic reads. SPARSE balances existing biased reference databases by grouping reference genomes into similarity-based hierarchical clusters, implemented as an efficient incremental data structure. SPARSE assigns reads to these clusters using a probabilistic model, which specifically penalizes non-specific mappings of reads from unknown sources and hence reduces false-positive assignments. Our evaluation on simulated datasets from two recent evaluation studies demonstrated the improved precision of SPARSE in comparison to other methods for species-level classification. In a third simulation, our method successfully differentiated multiple co-existing Escherichia coli strains from the same sample. In real archaeological datasets, SPARSE identified ancient pathogens with ≤ 0.02% abundance, consistent with published findings that required additional sequencing data. In these datasets, other methods either missed targeted pathogens or reported non-existent ones. SPARSE and all evaluation scripts are available at https://github.com/zheminzhou/SPARSE.

scholarly journals Antimicrobial-Loaded Bone Cement Does Not Negatively Influence Sonicate Fluid Culture Positivity for Diagnosis of Prosthetic Joint Infection

2016 ◽  
Vol 54 (6) ◽  
pp. 1656-1659 ◽  
Author(s):  
Kyung-Hwa Park ◽  
Kerryl E. Greenwood-Quaintance ◽  
Arlen D. Hanssen ◽  
Matthew P. Abdel ◽  
Robin Patel
Keyword(s):  
Bone Cement ◽  
Prosthetic Joint Infection ◽  
Joint Infection ◽  
Culture Positivity ◽  
Prosthetic Joint ◽  
Fluid Culture

We compared culture results to investigate the influence of antimicrobial-loaded cement on sonicate fluid culture positivity for the diagnosis of prosthetic joint infection. Fifty-four subjects were assessed. The sensitivities of sonicate fluid culture were 77.8% (14 of 18) in subjects with an antimicrobial-loaded cemented prosthesis and 58.3% (21 of 36) in subjects with an antimicrobial-free prosthesis.

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