Enhancing Sepsis Care: Expediting Blood Culture Processing for Rapid AST
September marked Sepsis Awareness Month and to support this important cause we are revisiting our previous post on rapid Antimicrobial Susceptibility Testing (rAST) from positive blood culture bottles (PBC). In that post, we discussed innovative assays, recently cleared by the FDA, that allow direct susceptibility testing from PBCs. While these all-in-one platforms are creative solutions to improve patient care, laboratories with limited resources say that the cost of new technology continues to be a significant hurdle. Fortunately, there are alternatives. For example, these labs can implement the Clinical and Laboratory Standards Institute (CLSI) blood culture direct rAST breakpoints, which we discuss further in this article, or bring in an FDA cleared positive blood culture processing system to expedite AST results, providing potential life-saving testing for patients.
FDA Cleared PBC Processing System
The eQUANT™ system, developed by Avails Medical, was designed to expedite positive blood culture processing for rAST by creating a 0.5 McFarland equivalent directly from PBC. The system utilizes an electronic biosensor to monitor changes in oxidation-reduction potential, enabling precise measurements of bacterial concentrations in blood culture. It generates a suspension equivalent to 0.5 McFarland standard by quantifying the bacterial load. The workflow of the assay takes three steps and requires about 5 minutes of hands-on time. An aliquot of the PBC is added to the assays proprietary eTUBE™ consumable, which is then added to their instrument. The sample is then ready in 70 minutes for downstream testing. A pilot study presented at ASM Microbe evaluated 59 PBCs and demonstrated the eQUANT system was accurate in inocula with 90% of bottles processed. When compared for performance on Microscan and Vitek, the authors observed 97.5% categorical agreement (CA), 0.8% very major error, 0.3% major error, and 2.1% minor errors. In February 2024 the eQUANT achieved FDA 510(k) clearance for Kirby-Bauer/Disk Diffusion with Gram-negative PBCs. Further applications may be available in the future.
The eQUANT system is an excellent alternative for laboratories to consider that wish to provide more rapid AST results and are unable to implement fully automated sample-to-AST testing methodologies, and desire fewer potential limitations than those with CLSI rAST workflow and breakpoints. It should also be noted that the company is leveraging their technology to develop their own automated platforms to go PBC-to-susceptibility.
CLSI rAST Breakpoints
Approximately 90% of PBC are monomicrobial, and by the time these cultures go positive the bacterial load reaches around 107-108 CFU/mL. These concentrations are comparable to a 0.5 McFarland standard, which is traditionally used for inoculating plates for antimicrobial susceptibility testing with Kirby Bauer Disks diffusion methods. In theory, a PBC could be directly plated onto agar, allowing AST to be performed with disk diffusion without the need for prior isolation of the organism. This workflow would remove the need to isolate the organism from the PBC and reduce AST reporting by 18-24 hours. In 2019, the European Committee of Antimicrobial Susceptibility Testing (EUCAST) came out with breakpoints for this testing, and it was followed up with CLSI breakpoints that allow testing directly from PBC and with incubation lengths of 8-10 hours and 16-18 hours.
Since the publication of these breakpoints, several laboratories have validated the breakpoints for their accuracy and utility. In July of 2024, Deng et al. evaluated the rAST testing of Enterobacterales vs broth microdilution. The study demonstrated 90% concordance at 10-hour time reads which met the VME and ME limits (≤1.5% and ≤3.0%) for all testing except for Enterobacter complex. They also identified 112 carbapenem-resistant pathogens from 314 isolates. Patient outcomes were not included in the analysis. Still, it would be expected that these data would lead to faster optimization of antibiotic treatment when supported by antimicrobial stewardship programs
There are notable limitations to the CLSI breakpoints for rAST, namely the narrow range of organisms covered for testing. Currently, breakpoints are available for a subset of organisms including Enterobacterales, P. aeruginosa and Acinetobacter spp, Haemophilus influenzae, Neisseria gonorrhea, S. aureus, and Streptococcus spp. While these organisms make up some of the more challenging organisms to treat due to various resistance mechanisms, less common pathogens are not yet available. Furthermore, the breakpoints focus on a limited selection of antibiotics that are the most crucial for managing sepsis.
Laboratories with experience and capacity to properly validate Kirby Bauer Disk Diffusion directly from PBC may utilize this method. However, for laboratories with limited resources, the FDA cleared solutions should be the preferred method.
Additional Resources
eQUANT™ system by Avails Medical
CLSI M100 Performance Standards for Antimicrobial Susceptibility Testing
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