Fractures, Bone, Infections
Conditions
Keywords
High throughput sequencing, Nanopore, Diagnosis, Trauma, Microbiome, Infection
Brief summary
Treatment of fracture related infection is challenging and often lead to failure in such situation that carry a high health cost burden. These infections are often polymicrobial, making the identification of all involved microorganisms a major concern to provide tailored antibiotic treatment. Culture-independent methods are needed to better represent the microbial diversity of infected wounds. Metagenomic sequencing might lead to an accurate microbiome characterization in infected trauma-related wound. Preliminary studies have reported results of metagenomic sequencing in diabetic foot infection but data focusing on non-diabetic infected patients are scarce. The impact of post-traumatic infected wound microbiome needs to be assessed, with regards to bacterial abundance, diversity including at the strain level and functional genes, along with their longitudinal evolution and association with clinical outcomes.
Detailed description
DNA will be extracted from samples carried out during surgical procedures. Different extraction protocols will be assessed to determine the best to be used for this type of tissue samples. Purified DNA will be quantified using the Qubit dsDNA High-Sensitivity Assay Kit (Invitrogen). The quality of the fragment length will be estimated with the DNA high-sensitivity kit in the 2100 Bioanalyzer (Agilent Technologies). DNA sequencing will be performed with Oxford Nanopore Technologie devices: MinIONTM and GridIONTM. Another sequencing of the same samples will be performed on the MiSeq after libraries preparation using the NexteraXT DNA Library Preparation Kit (Illumina). Sequenced OTU from both sequencing methods will be confronted at different taxonomic ranks and compared with conventional routine method results (bacterial culture). A functional analysis of sequences will be done to identify potential genes associated with clinical outcome.
Interventions
DIAGNOSTIC_TESTMetagenomic sequencing
Samples shall be submitted to high throughput sequencing using both illumine MiSeq and Oxford Nanopore Technologies.
Sponsors
Military Teaching Hospital Sainte Anne, Toulon
UMR Vitrome, IRD 257
Centre Hospitalier Intercommunal de Toulon La Seyne sur Mer
Study design
Observational model
COHORT
Time perspective
RETROSPECTIVE
Eligibility
Sex/Gender
ALL
Age
18 Years to No maximum
Inclusion criteria
* Age ≥ 18 years * Diagnosis of trauma-related infection
Exclusion criteria
* Participation in an interventional research during the study * Patient opposition * Absence of bone or soft tissue samples stored at -80°C
Design outcomes
Primary
| Measure | Time frame | Description |
|---|---|---|
| Microbiome of fracture-related and other trauma-related infection | About 2 months | Metagenomic sequencing will be used to determine the microbiome of trauma-related infections using Illumina MiSeq and Oxford Nanopore Technologies. Data will be compared with those from reference microbiological identification techniques (culture). |
Secondary
| Measure | Time frame | Description |
|---|---|---|
| Comparison of high throughput sequencing techniques yield | About 3 months | The FRI-associated microbiomes composition obtained with each sequencing methods will be compared in terms of abundance and variety (K coefficient and frequency) |
| Number and nature of virulence or resistance factors among identified OTU (operational Taxonomic Unit) | About 6 months | Functional annotation of sequenced bacterial genomes to assess the presence of virulence and/or resistance-associated factors, using Prokka software. |
| Number of bacteria and their relative abundance according to patients' outcome using the EBJIS (European Bone and Joint Infection) definition | About 6 months | To determine the association of the NGS-based microbiome longtitudinal composition in terms of variety (number of OTU) and abundance (number of reads) with infection persistence according to the EBJIS (European Bone and Joint Infection) definition. |
Countries
France
Outcome results
None listed