The time it takes to get results from a test for microbial identification—a significant variable in patient outcome that has historically been dependent on a blood culture procedure—has been dramatically shortened with the development of an entirely new approach.
In a study published in Nature, researchers at the Seoul National University and QuantaMatrix described an ultra-rapid antimicrobial susceptibility testing platform that can perform drug susceptibility profiling directly from the patient’s whole blood, eliminating the need for blood cultures. The researchers also offer a blood culture-free pathogen identification assay that is very sensitive and gives species information at every stage of its workflow, which is essential for interpreting antimicrobial susceptibility tests accurately. Compared to commercial, hospital-based antimicrobial susceptibility testing methods, this approach shortens a process that typically takes 48–72 hours to an average of about 13 hours (according to simulations), potentially saving countless lives and preventing the rise of antimicrobial resistance.
Blood culture-free, ultra-rapid antimicrobial susceptibility testing
Although there have been numerous reports of blood-based bacterial isolation methods, there have been minimal successful attempts to incorporate these into antimicrobial susceptibility testing. This is due to the inability to isolate rare microorganisms from large volumes of complex patient specimens and the limited breadth of pathogenic coverage. While other diseases have known invading pathogens based on their physical properties or surface composition (such as protein, lipid, or carbohydrate types), bacteremia often has unknown invading pathogens. Producing them alive with classical antibodies, aptamers, or nanobodies makes it difficult. The researchers used a peptide called sβ2GPI, which comes from a protein in human innate immunity that can effectively identify common patterns shared by different pathogens. Isolating pathogens from whole blood using sβ2GPI peptides allows for real-time monitoring of growth in a purified medium and reduces microbial expansion.
To confirm the presence of and discriminate pathogenic types, the researchers created an assay consisting of a library of silica-coated micro-discs (diameter 50 µm), each immobilized with single-stranded DNA probes (5′-amine modified) on their carboxylated surface, which can hybridize genomic sequences of the corresponding pathogen. First, resuspended pathogens are lysed for DNA extraction, amplification, and biotin labeling. Amplicons are then mixed with microdiscs functionalized with DNA detection probes targeting bacteria-specific ID and resistance genes. Finally, the resulting product is fluorescently labeled for imaging and subsequent analysis. The researchers also developed an ultra-rapid antimicrobial susceptibility testing chip to reduce turnaround time. This chip enables the calculation of drug susceptibility with just a few microbial cells. This expedites the rapid culture process that follows the isolation of blood-borne pathogens by saving multiple doubling periods.
The authors admitted that one limitation of the technique is that attempts to distinguish between non-growing persisters and dead pathogens after antibiotic evaluation should be pursued because the absence of growth does not always indicate susceptibility, a limitation shared by all current phenotype-based antimicrobial susceptibility tests. To ensure the method’s effectiveness and clinical value, future studies should validate it in a more diverse group of patients. Additionally, there should be efforts to automate the system and reduce costs through mass production, as the substantial (non-technical) delay caused by laboratory working hours must be addressed. Nevertheless, this approach may usher in a new era of emphasis on creating next-generation platforms for antimicrobial susceptibility testing that do not require blood cultures, thereby revolutionizing the present state of bacteremia diagnostics.
