Microbial analysis without cell purification
The invention provides systems and methods for rapid automated identification of microbes and antimicrobial susceptibility testing (AST) directly from a patient specimen, without specimen preparation. Specimens are loaded into an analytical cartridge for processing. Analytical cartridges are preloaded with species-specific labels that are used to identify and enumerate microbes in the specimen. Instruments, such as analyzers can be used to interact with analytical cartridges to carry out methods of the invention all within the cartridge.
1 . A method for antimicrobial susceptibility testing, the method comprising:
obtaining a polymicrobial specimen;
dividing the specimen into division wells wherein at least some of the division wells comprise different antimicrobial agents or concentrations of one or more agents;
incubating the specimen in the wells to allow differential growth in response to the different antimicrobial agents or concentrations of antimicrobial agents;
labeling specific target cells in the specimen with photonically fluorescently labeled nucleic acid probes wherein said fluorescently labeled nucleic acid probes bind to target specific sequences on ribosomal RNA;
incubating said labeled target cells with magnetic particles thus forming complexes of fluorescently labeled target cells bound to magnetic particles, wherein said photonic fluorescent labeling of said target cells and said incubating of said target cells with magnetic particles occur in a concerted reaction;
depositing said complexes on a detection surface of each imaging well using magnetic force; and
counting individual target cells of the complexes in each imaging well using digital imaging to identify the antimicrobial agent or concentration of an antimicrobial agent that inhibits growth of the target cells.
2 . The method of claim 1 , in which one of said division wells is a no antibiotic control division well, wherein the no antibiotic control division well contains specimen, growth medium, and no antimicrobial agent.
3 . The method of claim 2 , further comprising comparing a number of cells obtained from the no-antibiotic control division well to the number of cells obtained from the division wells containing specimen, growth medium and said antimicrobial agents to identify the antimicrobial agent or concentration of antimicrobial agent that inhibits growth of the target cells.
4 . The method of claim 1 , wherein the incubating step includes fluorescently labeling the microbes in a target cell specific manner, and the counting step includes imaging each well and counting fluorescent spots in an image.
5 . The method of claim 1 , wherein the method does not comprise nucleic acid amplification.
6 . The method of claim 1 , wherein the labeling step lasts less than an hour and is performed at a temperature lower than 40 degrees C. in a growth medium.
7 . The method of claim 1 , wherein the dividing, incubating, and counting steps are performed within a single cartridge.
8 . The method of claim 7 , further comprising:
loading the cartridge into an analyzer, wherein the analyzer uses a one or more magnet to separate the target cell from other parts of the specimen and uses an imaging subsystem to perform the counting step.
9 . The method of claim 8 ,
wherein the analyzer uses a pneumatic subsystem to perform the dividing step within the cartridge,
wherein the division wells are within the cartridge and are preloaded with the different agents or concentrations of antimicrobial agents,
wherein, after the dividing and the incubating steps, the analyzer transfers contents of the division wells to corresponding reagent wells to therein expose the incubated specimen to the target cell binding magnetic beads and to target cell specific detectable labels.
10 . The method of claim 8 , wherein the analyzer uses a magnet to pull complexes of photonically labeled target cells bound to magnetic particles through a dye cushion so that they are deposited on the detection surfaces of each imaging well within the cartridge, wherein said dye cushion attenuates the imaging background arising from photonically labeled probe molecules not bound to said deposited complexes.
11 . The method of claim 10 , wherein the magnet pulls the microbe binding magnetic beads through a dye cushion that excludes unbound detectable labels from the detection surface.
12 . The method of claim 8 , wherein the analyzer uses a carousel and/or a mechanical cartridge conveyance to transfer the cartridge to an imaging subsystem to perform the counting step.
13 . The method of claim 1 , wherein the incubating and counting steps accomplish fluorescent in situ hybridization (FISH) analysis substantially at physiological temperature within the analyzer.
14 . The method of claim 1 , wherein identifying the antimicrobial agent that inhibits growth of the microbe comprises comparing the number of complexes in the incubated specimen to the number of complexes in an unincubated specimen.
15 . The method of claim 8 , wherein the analyzer uses the cartridge to perform target cell specific counting of the individual microbes after the differential growth.
16 . The method of claim 8 , wherein the analyzer manipulates the cartridge to perform the dividing, incubating, and counting steps.
17 . The method of claim 1 , wherein the specimen does not require any chemical or molecular sample preparation techniques by a user before dividing the specimen into division wells.
18 . The method of claim 1 , wherein the specimen is selected from the group consisting of: whole blood, plasma, serum, urine, sputum, stool, wound, peritoneal fluid, pus, lymph, vaginal secretions, nasal secretions, and tissue specimens.