Clinical Metagenomic of Post-traumatic Infections

Study Overview
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Brief Summary
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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
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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.

Official Title
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Clinical Metagenomic Next-Generation Sequencing for Microbial Infections in Trauma

Conditions
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Fractures, Bone, Infections

Intervention / Treatment
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* Diagnostic Test: Metagenomic sequencing


Participation Criteria
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Eligibility Criteria
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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

Ages Eligible for Study
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Minimum Age: 18 Years

Sexes Eligible for Study
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All

Study Plan
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How is the study designed?
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Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Trauma related infection<br> | Diagnostic Test: Metagenomic sequencing<br>* Samples shall be submitted to high throughput sequencing using both illumine MiSeq and Oxford Nanopore Technologies.<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Microbiome of fracture-related and other trauma-related infection | 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). | About 2 months | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Comparison of high throughput sequencing techniques yield | The FRI-associated microbiomes composition obtained with each sequencing methods will be compared in terms of abundance and variety (K coefficient and frequency) | About 3 months | 
| Number and nature of virulence or resistance factors among identified OTU (operational Taxonomic Unit) | Functional annotation of sequenced bacterial genomes to assess the presence of virulence and/or resistance-associated factors, using Prokka software. | About 6 months | 
| Number of bacteria and their relative abundance according to patients' outcome using the EBJIS (European Bone and Joint Infection) definition | 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. | About 6 months | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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High throughput sequencing, Nanopore, Diagnosis, Trauma, Microbiome, Infection