Assays for improving automated antimicrobial susceptibility testing accuracy
Inventors
STERN, Eric • Flentie, Kelly • Phelan, Nicholas
Assignees
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Publication Number
US-11835523-B2
Publication Date
2023-12-05
Expiration Date
Abstract
Phenotypic antimicrobial susceptibility testing (AST), the gold-standard diagnostic that indicates whether an antimicrobial will be clinically effective, often suffer the slowest times-to-result for the most resistant pathogens. Here we introduce novel assays to be performed in parallel with standard AST assays that enable rapid, same-shift reporting of AST results for a plurality of pathogens. The assays developed here are further capable of detecting resistance to carbapenems, the most powerful class of beta-lactams commonly used as “last-resort” antimicrobials.
Core Innovation
The invention provides a method for performing automated antimicrobial susceptibility testing of a carbapenem antimicrobial by running a dilution assay and a carbapenemase assay in parallel. The dilution assay inoculates a microbial inoculum of concentration C0 into a plurality of fluid wells defining a dilution series for the carbapenem antimicrobial and measures a signal associated with microbial growth in each well. The method thereby generates dilution-assay data for defining and labeling a microorganism as carbapenem susceptible or carbapenem resistant.
In parallel, the method performs a carbapenemase assay by measuring a signal associated with carbapenem degradation in a first well comprising the carbapenem antimicrobial and a microbial inoculum with concentration CR, where CR>10×C0. The method also measures signals associated with carbapenem degradation in one or more second wells having different contents than the first well so that they act as controls for the first well. The method compares the signals measured from the carbapenemase assay wells.
The invention then combines the data derived from the dilution assay with that derived from the carbapenemase assay to define and label the microorganism as carbapenem susceptible or carbapenem resistant. The disclosed architecture further integrates an automated antimicrobial susceptibility test assay cartridge that includes antimicrobial dilution series wells together with a series of assay wells for a carbapenemase assay probe, where the carbapenemase assay results influence algorithmic MIC determinations.
Claims Coverage
The provided excerpt contains three independent claims. Across these independent claims, the inventive features are centered on parallel dilution-growth readouts with carbapenem-degradation signals, cartridge well architecture for probe and inhibitor controls, and using carbapenemase assay results to influence algorithmic MIC determinations.
Parallel automated dilution and carbapenemase assays for MIC labeling
Perform an automated antimicrobial susceptibility testing method for a carbapenem antimicrobial by performing a dilution assay that inoculates a microbial inoculum of concentration C0 into a plurality of fluid wells defining a dilution series and measuring signals associated with microbial growth, while in parallel performing a carbapenemase assay that measures signals associated with carbapenem degradation in a first well with concentration CR where CR>10×C0 and in one or more second control wells with different contents; compare the carbapenemase signals and combine dilution-assay data with carbapenemase-assay data to define and label the microorganism as carbapenem susceptible or carbapenem resistant.
Cartridge architecture with beta-lactam dilution series and beta-lactamase probe with inhibitor wells
Provide an automated antimicrobial susceptibility test assay cartridge for a beta-lactam/beta-lactamase inhibitor antimicrobial comprising two or more assay wells including an antimicrobial dilution series for a beta-lactam antimicrobial inoculated at concentration C0 with a microbial inoculum, and a series of assay wells for a beta-lactamase inhibitor assay probe inoculated at concentration CR from the same microbial inoculum, including one or more assay wells comprising a beta-lactamase probe and two or more assay wells comprising the same beta-lactamase probe and a beta-lactamase inhibitor at one or more concentrations.
Cartridge for carbapenems with carbapenemase assay using beta-lactamase probe and specific inhibitors that influence algorithmic MIC
Provide an automated antimicrobial susceptibility test assay cartridge for one or more carbapenems comprising two or more assay wells comprising an antimicrobial dilution series for a carbapenem antimicrobial, and a series of assay wells for a carbapenemase assay comprising one or more assay wells comprising a beta-lactamase probe and two or more assay wells comprising the same beta-lactamase probe and two or more inhibitors selected from the list of clavulanate, cloxacillin, and tazobactam, wherein the cartridge is adapted for inoculation with microorganisms from a single microbial inoculum and the results of the carbapenemase assay influence algorithmic MIC determinations of one or more carbapenems.
Cartridge for carbapenems using saline, ionic zinc, and pH indicator carbapenemase wells that influence algorithmic MIC
Provide an automated antimicrobial susceptibility test assay cartridge for one or more carbapenems and/or beta-lactam/beta-lactamase inhibitors comprising two or more assay wells including an antimicrobial dilution series for a carbapenem antimicrobial and a series of assay wells for a carbapenemase assay comprising one or more assay wells comprising saline, a carbapenem, ionic zinc, and a pH indicator, one or more assay wells comprising saline, a carbapenem, ionic zinc, a pH indicator, and a microbe sample comprising <1×10^8 CFU intact microbes, one or more assay wells comprising saline, ionic zinc, and a pH indicator, and one or more assay wells comprising saline, ionic zinc, a pH indicator, and a microbe sample comprising <1×10^8 CFU intact microbes, wherein the cartridge is adapted for inoculation with microorganisms from a single microbial inoculum and wherein the results of the carbapenemase assay influence algorithmic MIC determinations of one or more carbapenems.
Across the independent claims, the coverage emphasizes integrated assay design: a dilution assay measuring signals associated with microbial growth and a carbapenemase assay measuring signals associated with carbapenem degradation using control wells and specified probe, inhibitor, or saline, ionic zinc, and pH indicator compositions, with carbapenemase assay results combined with or used to influence algorithmic MIC determinations for carbapenem susceptible versus carbapenem resistant labeling.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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