TARGETED SEQUENCING TOWARD PRECISION WOUND CARE

Background: Genomics and informatics analyses continue to advance at an accelerating pace and, in the modern age of precision medicine, will prove invaluable for exploring the role of microbial communities in healthy and disease states. These technologies hold potential for tremendous benefit in combat casualty care. The severely invasive nature of combat trauma creates massive regions of injury and infection, requiring specialized diagnostic and aggressive therapeutic approaches. Even in the hands of experienced surgeons, some wounds fail to heal due to a lack of actionable diagnostic information. More timely, informative evaluation of wound infection would reduce morbidity, shorten hospital stays, and improve rehabilitation for combat-wounded Service members. Microbial metagenomic sequencing has the potential to provide this information through analysis of genomic factors such as antibiotic resistance; however, whole genome sequencing does not yield the coverage required for sensitively detecting and analyzing individual microbial genes due to background created by human genomic sequence in clinical samples. A new approach is needed to bring the utility of DNA sequencing to management of wound infections. The low limit of detection and the high specificity that could be delivered by an approach employing targeted sequencing would facilitate detection of microbial genomic determinants in clinical samples, even before they result in emergent phenotypes that are detrimental to patient care.Objective: To develop and validate a targeted sequencing platform designed to selectively sequence thousands of microbial genomic regions relevant to wound infection and healing, thereby facilitating high confidence detection of critical microbial factors that are otherwise difficult or impossible to assess. Microbial factors will include species identity, antimicrobial resistance, and virulence/biofilm formation.Specific Aims: (1) Select microbial genomic targets and construct targeted sequencing panel. (2) Validate sensitivity and specificity of targeted sequencing panel using reference genome controls. (3) Validate use of targeted sequencing panel for predicting select clinical parameters in combat injuries.Study Design: Microbial genes with an anticipated role in wound progression will be selected from multiple sources, including data previously collected by the proposed team, previous experience developing detection panels for antimicrobial resistance, and existing genomic databases for resistance and virulence. Bioinformatics design will be applied for optimization of oligonucleotide sequences for pre-amplification/enrichment. For validating sensitivity and specificity of the panel, reference genomes containing regions targeted in the designed panel will be selected for testing. These genomes, in isolation and in combination, will be spiked into a human background at defined genome copy numbers. The targeted panel will be applied to these samples, and detection of the targeted reference genome regions will be verified and measured at each spiked copy number. To validate utility in combat injury diagnostics, the panel will then be applied to extremity wound samples collected from Service member patients. Sequence detection of the targeted regions will be examined for association with select clinical outcomes, including healing status, microbial colonization, and antimicrobial regimens administered.Innovation and Military Impact: The use of targeted sequencing applied to polymicrobial populations in wound infections, as proposed here, has not been previously explored, and such output has never been evaluated for clinical decision support in combat injury care. Targeted sequencing enables gene- and mutation-level analysis in near point-of-care applications, which could potentially change the landscape of patient care outcomes. Combat trauma is by nature intensely invasive and would benefit tremendously from the proposed novel approach, which could guide precise interventions applied on an individual patient level. Targeted sequencing technologies have a world of possible applications yet unexplored, and this effort presents an outstanding opportunity for carrying this benefit to precision military medicine.