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A "Culture" Shift: Broad Bacterial Detection, Identification, and Antimicrobial Susceptibility Testing Directly from Whole Blood.

Clinical Chemistry 2018 October
BACKGROUND: The time required for bloodstream pathogen detection, identification (ID), and antimicrobial susceptibility testing (AST) does not satisfy the acute needs of disease management. Conventional methods take up to 3 days for ID and AST. Molecular diagnostics have reduced times for ID, but their promise to supplant culture is unmet because AST times remain slow. We developed a combined quantitative PCR (qPCR)-based ID+AST assay with sequential detection, ID, and AST of leading nosocomial bacterial pathogens.

METHODS: ID+AST was performed on whole blood samples by ( a ) removing blood cells, ( b ) brief bacterial enrichment, ( c ) bacterial detection and ID, and ( d ) species-specific antimicrobial treatment. Broad-spectrum qPCR of the internal transcribed spacer between the 16S and 23S was amplified for detection. High-resolution melting identified the species with a curve classifier. AST was enabled by Ct differences between treated and untreated samples.

RESULTS: A detection limit of 1 CFU/mL was achieved for Acinetobacter baumannii , Escherichia coli , Klebsiella pneumoniae , and Staphylococcus aureus . All species were accurately identified by unique melting curves. Antimicrobial minimum inhibitory concentrations were identified with Ct differences of ≥1 cycle. Using an RNA target allowed reduction of AST incubation time from 60 min to 5 min. Rapid-cycle amplification reduced qPCR times by 83% to 30 min.

CONCLUSIONS: Combined, sequential ID+AST protocols allow rapid and reliable detection, ID, and AST for the diagnosis of bloodstream infections, enabling conversion of empiric to targeted therapy by the second dose of antimicrobials.

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