Highly multiplexed targeted sequencing strategy for infectious disease surveillance

Area of Science:

• Microbiology
• Genomics
• Infectious Disease Diagnostics

Background:

• Characterizing diseases of poverty is challenging due to a lack of affordable and comprehensive detection platforms.
• Next-generation sequencing (NGS) offers high throughput but is limited by low microbial DNA concentrations and complex preparation/analysis.
• Existing methods hinder efficient disease control and resource allocation in global health initiatives.

Purpose of the Study:

• To adapt molecular inversion probes (MIPs) for cost-effective target enrichment in microbial infection characterization.
• To develop a targeted sequencing approach for identifying pathogens and antimicrobial resistance markers (AMRs) in clinical samples.
• To simplify infectious disease surveillance in medium- to low-resource settings.

Main Methods:

• Designed a molecular inversion probe (MIP) panel targeting bacterial genera, species, fungi, and antimicrobial resistance markers (AMRs).
• Utilized short-read sequencing for targeted DNA enrichment from blood samples.
• Adapted MIPs for high specificity and throughput detection of microbial targets.

Main Results:

• The MIP approach demonstrated high specificity, detecting pathogen DNA at concentrations as low as 1 in 1000 targets within host DNA.
• Successfully identified pathogens and AMRs in 20 out of 24 sepsis patient samples previously analyzed by blood culture.
• The assay provides accurate profiling of microbial infections and resistance markers.

Conclusions:

• The targeted MIP assay offers high-throughput identification of microbial pathogens and AMRs with excellent specificity.
• The simplified workflow, requiring minimal sample preparation and bioinformatics, makes it suitable for resource-limited settings.
• This approach enhances the characterization and surveillance of infectious diseases globally.