INSTRUMENT AND SYSTEM FOR RAPID MICROORGANISM IDENTIFICATION AND ANTIMICROBIAL AGENT SUSCEPTIBILITY TESTING

• US 20160289729A1
• Filed: 03/30/2016
• Published: 10/06/2016
• Est. Priority Date: 03/30/2015
• Status: Active Grant

• Current Assignee: Accelerate Diagnostics, Inc.
• Original Assignee: Accelerate Diagnostics, Inc.
• Inventors: Ketterer, Matthew, Harrison, Josh, Cote, Jasmin, Hance, Ken, Mensack, Meghan, Michel, Carlos, Allers, Elke, Gamage, Dulini, Prisbrey, Landon, Gusyatin, Oleg, Turng, Ben, Richards, William L., Ashby, Austin, Marshall, Kevin, Mette, Matthew, Richards, Paul, Showalter, Wayne, Halbert, Phillip C., Bourgeois, Solene, Metzger, Steven W., Shamsheyeva, Alena, Ghusson, Andrew, Reinhardt, Kurt
• Granted Patent: US 10,023,895 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: B01L 2300/0893 having a very large number of wells, microfabricated wells
• CPC Class Codes: B01L 3/527 for a plurality of reagents
• CPC Class Codes: C12Q 1/025 for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics antimicrobial activity C12Q1/18
• CPC Class Codes: C12Q 1/04 Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor C12Q1/6897 takes precedence
• CPC Class Codes: G01N 1/38 Diluting, dispersing or mixing samples
• CPC Class Codes: G01N 15/06 Investigating concentration of particle suspensions by weighing G01N5/00; investigating sedimentation of particle suspensions G01N15/04; investigating individual particles G01N15/10
• CPC Class Codes: G01N 15/075 by optical means
• CPC Class Codes: G01N 2021/6419 Excitation at two or more wavelengths
• CPC Class Codes: G01N 2035/00346 Heating or cooling arrangements
• CPC Class Codes: G01N 2035/0443 for reagents
• CPC Class Codes: G01N 2035/0444 for cuvettes or reaction vessels
• CPC Class Codes: G01N 2035/0446 Combinations of the above
• CPC Class Codes: G01N 2035/0455 Coaxial carousels
• CPC Class Codes: G01N 2035/1032 Dilution or aliquotting
• CPC Class Codes: G01N 21/6458 Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16
• CPC Class Codes: G01N 35/025 having a carousel or turntable for reaction cells or cuvettes
• CPC Class Codes: G01N 35/1002 Reagent dispensers
• CPC Class Codes: G02B 7/28 Systems for automatic generation of focusing signals

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.

Claims

1. A system for automated microorganism identification and antibiotic susceptibility testing comprising:

a reagent cartridge comprising a plurality of wells;

a reagent stage wherein the reagent stage comprises an annular shape defining an interior opening, wherein the reagent stage is configured to rotate in a first plane and wherein the reagent stage is configured to receive the reagent cartridge;

a cassette comprising a plurality of microfluidic sample channels structured to receive samples, each of the plurality of microfluidic sample channels comprising an inlet port configured to receive a pipette tip;

a cassette stage located within the interior opening of the reagent stage, wherein the cassette stage is configured to rotate and move laterally in the first plane, and wherein the cassette stage is configured to receive the cassette;

a pipettor assembly configured to move a plurality of reagents between the plurality of wells of the reagent cartridge and the inlet ports of each of the plurality of microfluidic sample channels;

an optical detection system configured to obtain darkfield and fluorescence photomicrographs of one or more microorganisms contained in the plurality of microfluidic sample channels; and

a controller configured to direct operation of the system and process microorganism information derived from photomicrographs obtained by the optical detection system.

2. The system according to claim 1, wherein the plurality of microfluidic sample channels are arranged in a radial configuration about a center of the cassette, and wherein each microfluidic sample channel comprises one or more of an outlet port and an inlet port.

21. A method of identifying microorganisms in a patient sample comprising:

introducing a patient sample to the plurality of microfluidic channels of the cassette of the system of claim 1;

introducing one or more labeled universal control probes and one or more labeled target probes that recognize and bind sequences of microorganisms;

incubating the labeled probes with the sample for a time sufficient to enable binding of the probes to microorganisms in the patient sample;

capturing images of multiple fields of view of one or more microorganisms bound to labeled probes;

performing morphokinetic analysis to identify characteristics of one or more imaged microorganisms;

inputting data from the morphokinetic analysis to a probability expectation model of distribution to identify one or more microorganisms in the patient sample, wherein identification of pathogens is based on a combination of posterior probabilities from multiple microfluidic channels; and

matching a signal pattern of an unknown microorganism with a posterior probability density function of a labeled target probe using an empirical threshold value.

22. The method of claim 21, wherein the patient sample is polymicrobial.

3. A reagent cartridge kit for use in an automated microorganism identification and antibiotic susceptibility testing system, comprising:

a reagent cartridge comprising a plurality of wells and defining an opening;

a cassette configured to fit over or in the opening in the reagent cartridge; and

a seal shaped to cover at least portions of the cassette and the reagent cartridge.

4. The reagent cartridge kit of claim 3, further comprising a cassette holder shaped to receive the cassette, the cassette holder having an outer dimension shaped for coupling the cassette holder over or in the opening in the reagent cartridge.

5. A method for focusing on a microorganism to be imaged, comprising:

providing prescan information wherein intensity weighting versus focus position is obtained for a plurality of focus positions of an objective;

projecting light from a light source through the objective at an image plane;

capturing a first image of reflected light with the objective at a first position;

capturing a second image of reflected light with the objective at a second position;

computing intensity weightings of the reflected light for the first and second images;

calculating a focus position using the computed intensity weightings in association with the prescan information;

moving the objective to the calculated focus position; and

capturing an image of a microorganism with the objective in the calculated focus position.

6. The method of claim 5, wherein the light source is a first light source used for generating a reflection off of a glass and liquid interface at the image plane and wherein the capturing of the image is with a second light source.

7. The method of claim 5, wherein the first position is an out-of-focus position a first fixed distance from a first side of the 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.

8. The method of claim 5, further including computing a center of the reflected light in the first image and comparing the center to a known center to determine an offset distance from the known center and only using the first image if the offset distance is within a predetermined limit.

9. A system for autofocusing on a microorganism to be imaged, comprising:

a first light source for projecting a beam of light and generating a reflective spot on an image plane;

a second light source for projecting light to capture an image of the microorganism on the image plane;

a movable objective positioned between the first and second light sources and the image plane for allowing focusing on the image plane;

a camera for capturing the reflective spot and for capturing the image of the microorganism using the objective to focus; and

a controller for receiving two or more images of the reflective spot from the camera and for modifying a position of the objective based on intensity weightings of the reflective spots.

10. The system of claim 9, wherein the intensity weightings relate to a greyscale value associated with each pixel that is weighted according to each pixel'"'"'s position relative to a center.

11. A computer-readable storage medium including instructions, which upon execution, cause a computer system to perform autofocus operations, comprising:

move an objective using a control board to a first position so that a focus point is a predetermined distance in front of an image plane;

project a first light source through the objective, the light source designed to generate a first spot-shaped reflection off of the image plane;

capture a first image of the first spot-shaped reflection;

move the objective to a second position so that the focus point is a predetermined distance behind the image plane;

project the first light source through the objective to generate a second spot-shaped reflection;

capture a second image of the second spot-shaped reflection; and

perform intensity weighting analysis on the first and second images and using the intensity weighting analysis to determine a focus position for the objective.

12. A system comprising:

a system controller comprising;

a processor; and

a computer-readable storage medium storing instructions that, when executed by the processor, cause the system controller to perform operations comprising;

extracting three or more subsamples from a sample,

diluting each subsample using a distinct dilution factor,

determining a cell concentration of each diluted subsample,

using the cell concentrations and the distinct dilution factors to estimate a non-linear effective dilution curve for the sample,

determining a target dilution factor to dilute the sample to a target cell concentration using the non-linear effective dilution curve, and

diluting a remainder of the sample using the determined target dilution factor.

13. The system of claim 12, further comprising:

an instrument pipettor; and

wherein the extracting the three or more subsamples comprises using the instrument pipettor to remove each subsample from the sample.

14. A method for dynamically diluting a sample to a target cell concentration, the method comprising:

determining, using a processor, a target dilution factor, the determining comprising;

identifying a growth factor associated with a microorganism in the sample;

diluting one or more subsamples of a sample using different sample dilution factors,

for each of the one or more diluted subsamples, determining a cell concentration of the diluted subsample,

creating a model of a non-linear dilution curve using the one or more determined cell concentrations and the associated sample dilution factors, and

determining the target dilution factor using the model of the non-linear dilution curve, the growth factor, and the target cell concentration; and

diluting the sample using the determined target dilution factor.

15. The method of claim 14, wherein determining the target dilution factor comprises diluting three or more subsamples of the sample using different dilution factors and creating a model of the non-linear dilution curve using the three or more determined cell concentrations form the three or more subsamples.

16. The method of claim 15, wherein creating the model of the non-linear dilution curve comprises using the following formula:

17. A method of controlling temperature in a system, comprising:

reading an air temperature in an enclosure from a temperature sensor;

determining that the air temperature of the enclosure is to be increased;

determining, using a processor, components in the system that are currently idle; and

increasing power to the determined components so that they generate heat, while the determined components remain idle.

18. The method of claim 17, wherein the determined components are two or more stepper motors that have an active mode wherein the stepper motors cause movement within the system and an idle mode wherein the stepper motors are not active and maintain a current position.

19. A computer-readable storage medium including instructions, which upon execution, cause a computer system to control temperature in an enclosure, comprising:

read a temperature sensor to obtain a current temperature within the enclosure;

determine a level of cooling of air within the enclosure is needed;

for components within the system that are idle, decrease power to the components to minimize heat generation; and

control fan speed, using a processor, based on the determined level of cooling needed.

20. The computer-readable storage medium of claim 19, further including determining a level of heating required within the enclosure and increasing power to the components that are idle in order to generate heat.

23. A method of identifying a target microorganism, comprising:

capturing, in a camera, multiple images of an object using different modalities of light;

determining features of the objects identified;

providing a plurality of models based on signals, noise and cross-talk for known microorganisms;

calculating a probability distribution for a signal and at least one of noise or cross-talk using the plurality of models and the determined features of the objects identified; and

determining whether or not the microorganism is present in at least one of the captured multiple images using the calculated probability distribution.

24. The method of claim 23, wherein the capturing of the multiple images includes using at least two fluorescent light sources and at least one light source adapted for dark-field imaging.

25. A computer-readable storage medium including instructions, which upon execution, cause a computer system to identify a microorganism, comprising:

in a camera, capture a dark field image and at least one fluorescent image of an object of interest;

identify, using a processor, multiple features associated with the object;

compare the multiple features to a model that includes signal, noise and cross-talk data for a microorganism; and

determine whether or not the object of interest is the microorganism.

26. The computer-readable storage medium of claim 25, wherein the at least one fluorescent image is a first image using a first fluorescent light source at a first wavelength and further including instructions that capture an image of the object of interest using a second fluorescent light source at a second wavelength.

27. A reagent cartridge comprising:

a plurality of wells comprising reagents; and

an opening,

wherein the plurality of wells comprising reagents comprise at least one well comprising a probe and at least one well comprising an antimicrobial agent.

28. The reagent cartridge of claim 27, wherein the at least one well comprising a probe comprises a well comprising a Staphylococcus aureus probe, a well comprising a coagulase-negative staphylococci probe, a well comprising a Staphylococcus lugdunensis probe, a well comprising an Enterococcus faecalis probe, a well comprising an Enterococcus faecium probe, a well comprising a Streptococcus agalactiae probe, a well comprising a Streptococcus spp. probe, a well comprising an Escherichia coli probe, a well comprising a Klebsiella spp. probe, a well comprising an Enterobacter spp. probe, a well comprising a Citrobacter spp. probe, a well comprising a Proteus spp. probe, a well comprising a Serratia marcescens probe, a well comprising a Pseudomonas aeruginosa probe, a well comprising an Acinetobacter baumannii probe, a well comprising a Candida albicans probe, and a well comprising a Candida glabrata probe.

29. A cassette integrated into an automated microorganism identification and antibiotic susceptibility testing system comprising:

a glass support adhered to a molded polymer disc; and

a plurality of microfluidic channels arranged in a radial configuration across the cassette.