Instrument and system for rapid microorganism identification and antimicrobial agent susceptibility testing
Inventors
Ashby, Austin • Showalter, Wayne • Cote, Jasmin
Assignees
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Publication Number
US-10619180-B2
Publication Date
2020-04-14
Expiration Date
Abstract
A system for automated microorganism identification and antibiotic susceptibility testing comprising a reagent cartridge, a reagent stage, a cassette, a cassette, stage, a pipettor assembly, an optical detection system, and a controller is disclosed. The system is designed to dynamically adjust motor idle torque to control heat load and employs a fast focus process for determining the true focus position of an individual microorganism. The system also may quantify the relative abundance of viable microorganisms in a sample using dynamic dilution, and facilitate growth of microorganisms in customized media for rapid, accurate antimicrobial susceptibility testing.
Core Innovation
The invention relates to a method for focusing on a microorganism to be imaged using prescan information that includes intensity weighting versus focus position for a plurality of focus positions of an objective. Light is projected through the objective to an image plane, and reflected light is captured at a first objective position and at a second objective position, the first and second objective positions being out-of-focus positions located at fixed distances from opposite sides of the image plane.
The method computes intensity weightings of the reflected light for the first and second images and calculates a focus position by mapping the computed intensity weightings onto the prescan information. The objective is moved to the calculated focus position, and an image of a microorganism is captured with the objective in the calculated focus position to allow identification of the microorganism.
The approach supports calculations of intensity weightings using greyscale analysis and mapping onto prescan information, including prescan spot offset information versus focus position for multiple objective focus positions. The focus decision is further refined using image-based gating that compares a center of reflected light to a known center to determine an offset distance and optionally uses only the first image when the offset distance is within a predetermined limit.
The invention also relates to a dynamic dilution workflow for microbiology and antimicrobial susceptibility testing (AST) sample preparation that partitions a post-GEF sample into multiple subsamples and dilutes each subsample by a distinct dilution factor. Cell concentration for each subsample is determined, including by staining and counting, optionally after electrokinetic concentration, and the measured concentrations across multiple dilution test points are used to construct and fit or approximate a non-linear dilution curve.
Claims Coverage
The consolidated claims coverage includes two inventive methods from the inputs. One set covers a focusing method for a microorganism using prescan intensity-weighting information from opposite sides of an image plane, and the other covers a dynamic dilution workflow that determines a target dilution factor from measured cell concentrations across multiple dilution test points.
Mapping prescan intensity weightings to compute a focus position from opposite-side images
providing prescan information wherein intensity weighting versus focus position is obtained for a plurality of focus positions of an objective; computing intensity weightings of the reflected light for the first and second images; calculating a focus position by mapping the computed intensity weightings onto the prescan information, wherein the prescan information includes the intensity weighting versus focus position on both the first and second sides of the image plane
Opposite-side fixed-distance out-of-focus image capture for focusing
capturing a first image of reflected light with the objective at a first position and capturing a second image of reflected light with the objective at a second position, wherein the first position is an out-of-focus position a first fixed distance from a first side of an image plane and wherein the second position is an out-of-focus position a second fixed distance from a second side of the image plane, opposite the first side
Focusing workflow that moves the objective to the calculated focus position for microorganism identification
moving the objective to the calculated focus position; capturing an image of a microorganism with the objective in the calculated focus position, which allows an identification of the microorganism
Non-linear dilution curve for target dilution factor
partitioning a post-GEF sample into multiple subsamples and diluting each subsample by a distinct dilution factor; determining cell concentration for each subsample; using the measured concentrations across multiple dilution test points to construct and fit or approximate a non-linear dilution curve; and using the non-linear dilution curve to compute a target dilution factor for a desired target cell concentration
Growth-factor adjustment and validation logic
optionally adjusting for cell growth during the determination process and for the non-viable-cell fraction by using empirically determined growth factors per species, including growth-adjusted terms and validation and abort logic based on growth control
The consolidated inventive features comprise prescan-based focusing on opposite sides of an image plane for microorganism identification and a dynamic dilution workflow that derives a target dilution factor from a non-linear dilution curve, with optional growth-factor adjustment and validation logic.
Stated Advantages
Allows an identification of the microorganism by capturing an image with the objective in the calculated focus position.
Improved antimicrobial susceptibility testing (AST) results, including distinguishing true resistance from filamentation using nutrient-depleted Mueller-Hinton formulations.
Enables media selection for fastidious and delayed growers, including custom peptone-based media without blood for bacteria such as Serratia and Streptococcus.
Provides multiplexed automated microscopy/FISH ID and AST system performance results with reported sensitivity, specificity, and agreement with broth microdilution MICs.
Documented Applications
Microorganism identification using an automated focusing and imaging workflow based on prescan information and reflected-light images.
Nutrient-depleted Mueller-Hinton formulations to improve AST interpretation by distinguishing true resistance from filamentation.
Use of custom peptone-based media without blood for fastidious/delayed growers, including examples for Serratia and Streptococcus.
Multiplexed automated microscopy/FISH ID and AST system operation, with reported performance metrics such as sensitivity, specificity, and agreement with broth microdilution MICs.
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