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

Sample collection and processing.Intraoperative samples from the Nuffield Orthopaedic Centre (NOC) in Oxford University Hospitals (OUH), United Kingdom, between June 2013 and January 2017 were investigated. The NOC is a tertiary-level specialist musculoskeletal hospital, including a dedicated Bone Infection Unit, undertaking approximately 200 revision arthroplasties annually. A subset of samples submitted was chosen at random following culture to provide a ratio of approximately 2:1 bacterial culture-positive samples to culture-negative samples. For this study, no ethical review was required, because the study was a laboratory method development study focusing on bacterial DNA extracted from discarded samples identified only by laboratory numbers, with no personal or identifiable data. Sequencing reads identified as human on the basis of Kraken were counted and immediately permanently discarded.

Prosthetic joint implants and metalwork, received into the OUH microbiology laboratory following revision arthroplasty and operative management of other orthopedic device-related infection, were placed directly into single-use sterile polypropylene containers (Lock & Lock brand) and covered with between 10 ml and 400 ml of sterile 0.9% saline solution (Oxoid, Ltd., Basingstoke, United Kingdom) depending on the size of the prosthesis/device, with sufficient fluid to cover at least 90% of the prosthesis/device, up to a maximum of 400 ml. Sonication was performed as described previously (7) with minor modifications. Briefly, the implant was vortexed for 30 s, subjected to sonication for 1 min, followed by additional vortexing for 30 s. Sonication was performed in a Bransonic 5510 ultrasonic water bath (Branson, Danbury, CT, USA) at a frequency of 40 kHz. The resulting sonication fluid was plated in 0.1-ml aliquots onto Columbia blood agar (CBA) and chocolate agar plates (Oxoid, Ltd., Basingstoke, United Kingdom) for aerobic incubation and on CBA plates for anaerobic incubation. Aerobic incubation was performed at 35 to 37°C with 5% CO₂ for up to 5 days. Anaerobic incubation was performed at 35 to 37°C for 10 days. All cultured microorganisms were identified by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry on a Microflex LT using Biotyper, version 3.1 (Bruker Daltonics, Billerica, MA, USA). Samples were considered culture positive when growth of ≥50 CFU/ml was observed and additionally when growth of a highly pathogenic organism (including Staphylococcus aureus and Enterobacteriaceae) at <50 CFU/ml was observed.

Periprosthetic tissue samples were also collected during surgery, at the start of each procedure and using different surgical instruments for each sample, and processed by the microbiology laboratory. Briefly, Bactec bottles were inoculated with 0.5 ml of an inoculum generated by vortexing each tissue sample in 3 ml of 0.9% saline with sterile Ballotini balls for 15 s. Bottles were incubated under aerobic (Plus Aerobic/F culture vials) and anaerobic (Lytic/10 Anaerobic/F culture vials) conditions in a BD Bactec FX system (BD Biosciences, Sparks, MD, USA) for up to 10 days. Any bottles that flagged positive were subcultured onto agar plates and processed as described above to determine species.

Bacterial DNA extraction from sonication fluid.Prior to DNA extraction, sonication fluids were concentrated by centrifugation. Forty milliliters of fluid was transferred to a sterile, disposable 50-ml polypropylene tube and centrifuged at 15,000 × g in a Sorvall RC5C Plus centrifuge (SLA-1500 rotor with custom-made inserts) for 1 h at 16°C. Samples with a <40-ml starting volume of sonication fluid were made up to 40 ml with the same saline used for sonication. All but approximately 1 ml of the supernatant was discarded, and the pellet was resuspended in this volume of fluid before being passed through a 5-μm-pore-size syringe filter to deplete the number of human cells present and, therefore, the amount of human DNA in the final extract. Bacterial cells passing through the filter were pelleted, washed with 1 ml of 0.9% saline, and resuspended in 500 μl of molecular-biology-grade water before being mechanically lysed in Pathogen Lysis tubes (S) (Qiagen, Hilden, Germany) with a FastPrep 24 tissue homogenizer (MP Biomedicals, Santa Ana, CA, USA) (three times for 40 s at 6.5 m/s). DNA was extracted by ethanol precipitation, using GlycoBlue (Life Technologies, Paisley, UK) as a coprecipitant, and resuspended in 50 μl of 1× Tris-EDTA (TE) buffer. DNA was purified using AMPure XP solid-phase reversible immobilization (SPRI) beads (Beckman Coulter, High Wycombe, United Kingdom) and eluted in 26 μl of TE buffer. DNA concentration was measured using a Qubit 2.0 fluorometer (Life Technologies, Paisley, United Kingdom). A subset of samples was treated with an NEBNext microbiome DNA enrichment kit (New England BioLabs, Ipswich, MA, USA) for human DNA removal before an additional purification step using AMPure XP SPRI beads and final elution in 15 μl of TE buffer. Samples were extracted in batches, with a negative control of sterile 0.9% saline prepared alongside each batch using this same protocol.

Library preparation and Illumina MiSeq sequencing.DNA extracts quantified as ≥0.2 ng/μl were sequenced on a MiSeq desktop sequencer (Illumina, San Diego, CA, USA). Libraries were prepared as previously described, using a variation of the Illumina Nextera XT protocol (36). Briefly, 1 ng of DNA was prepared for sequencing following the Illumina Nextera XT protocol, with the modification of 15 cycles during the index PCR. Libraries were quantified using a Qubit 2.0 fluorometer, and their average sizes were determined with an Agilent 2200 TapeStation (Agilent Technologies, Santa Clara, CA, USA) before being manually normalized. Libraries were prepared and sequenced together in the same batch. Paired-end sequencing was performed using a 600-cycle MiSeq reagent kit (version 3), and samples were sequenced in batches of between 1 and 13 on a single flow cell.

Bioinformatics analysis.Raw sequencing reads were adapter trimmed using BBDuk (https://sourceforge.net/projects/bbmap/) and the adapter sequence file provided within the BBMap package; the following parameters were used: minlength, 36; k,19; ktrim, r; hdist, 1; mink, 12. Taxonomic classification of trimmed reads was performed using Kraken (37) and a bespoke database constructed from all bacterial genomes deposited in the NCBI RefSeq database as of January 2015 (updated January 2017 for the validation set; see below), with default parameters and no k-mer removals. Where no RefSeq genome was available for an organism cultured from a PJI at OUH since June 2013, available whole-genome assemblies were also added to the database where available in NCBI. Additionally, the Genome Reference Consortium Human genome build 38 (GRCh38) was included in the database to allow detection of host DNA. An optimum filtration threshold, using a Kraken filter that balanced false-positive removal and sensitivity, was determined using simulated data sets of reference genomes. Reference genomes representative of common pathogenic species were used to generate simulated Illumina MiSeq data sets and analyzed with Kraken using different filtration thresholds. A threshold value of 0.15 provided optimum read classification sensitivity while minimizing spurious results. Kraken output was visualized using Krona (38).

Statistical analysis.The performance of metagenomic sequencing was assessed by comparing the species identified from sequencing data with the species isolated from sonication fluid samples considered culture positive (i.e., ≥50 CFU/ml or growth of a highly pathogenic organism at <50 CFU/ml). In order to correct for samples which may contain small numbers of contaminating and nonspecific bacterial reads, a threshold was determined to identify the presence of true infection, using the first 50 samples sequenced as a derivation set. Two thresholds (1 and 2), and three parameters (a to c), were used to determine true infection: (i) samples with more reads from a given species than an upper-read cutoff (a) were included; (ii) samples with more species-specific reads than a lower-read cutoff (b) and with the percentage of species-specific reads as a proportion of all bacterial reads present above a percentage cutoff (c) were also included. Parameter values were selected by numerical optimization, using R, version 3.3.2, comparing sequencing results to sonication fluid culture results and maximizing the value of the Youden index (39) (sensitivity + specificity − 1). Sensitivity was calculated taking each species identified from each culture-positive sonication sample as a separate data point; thus, culture-negative samples did not contribute to the denominator, culture-positive samples with a single species contributed once, and culture-positive samples with two species contributed twice. Specificity was calculated using the total number of sonication samples as the denominator; as such samples contaminated by more than one species were counted as one false positive.

To ensure that read cutoff parameters were chosen without a penalty for potentially difficult to culture anaerobic species, the specificity value optimized was adjusted. Potential false-positive sequencing results with plausible fastidious anaerobic causes of infection (including Fusobacterium nucleatum, Propionibacterium acnes, and Veillonella parvula) in culture-negative samples were excluded when the specificity value used for parameter optimization was calculated.

Where bacterial reads were detected over the thresholds described above in a negative control, that sample was deemed to be contaminated. In the derivation set, in order to maximize the number of sequences available for analysis, only samples with evidence of the same contaminating organisms were excluded from each contaminated batch, rather than discarding the whole batch. During the derivation phase of the study, several batches of samples were found to be contaminated with DNA from other studies performed concurrently in the same research laboratory. Six of eight saline negative-control extracts displayed contamination with a single or multiple species at read numbers exceeding the determined diagnostic thresholds. All samples within these batches that displayed similar contamination levels were excluded from subsequent analysis if Kraken classification resulted in >100 reads corresponding to the majority of the contaminating species. A total of 22 samples (in addition to the 50 successfully sequenced) were excluded on this basis (Fig. 1). In batches 4 and 5 the negative controls were contaminated with Staphylococcus aureus, Escherichia coli, and P. acnes, and 15 samples were excluded with >100 reads from ≥2/3 species; in batch 6 the negative control was contaminated with Serratia marcescens, Klebsiella pneumoniae, E. coli, and P. acnes, and 2 samples with >100 reads from ≥3/4 species were excluded; in batches 2, 9, and 10 the negative control was contaminated with P. acnes, and 5 samples were excluded with >100 P. acnes reads. To address this issue, prior to the validation phase of the study, all pipettes, laminar flow and PCR hoods, and laboratory benches used for DNA extraction and library preparation were deep-cleaned with Virkon disinfectant and RNase Away surface decontaminant (Thermo Fisher Scientific, Waltham, MA, USA) in order to remove any possible sources of microbial or DNA contamination. All DNA extraction and library preparation reagents were replaced and used in preprepared per-batch aliquots used exclusively for this study. Sonication fluid samples were handled one at a time in the laminar flow hood, which was cleaned as above between each sample. Fresh gloves were worn each time a new sample was handled during the DNA extraction phase of the protocol. Having implemented these changes, for the validation phase, a more stringent quality control standard was applied, requiring the negative control to be contamination free for any of the samples in a batch to be analyzed.

Technical replicates.To ensure sequencing reproducibility, one DNA sample was sequenced twice, and biological replicates (DNA extraction process repeated) were sequenced for six samples (four in duplicate and two in triplicate).