Microbial analysis without cell purification

Abstract

The present invention provides a system and method for rapid, automated identification and antimicrobial susceptibility testing (AST) of microorganisms directly from patient specimens without specimen preparation. The specimen is loaded into an analytical cartridge for processing. The analytical cartridge is preloaded with species-specific labels used to identify and enumerate microorganisms in the specimen. An instrument, such as an analyzer, can be used to interact with the analytical cartridge to perform the method of the present invention entirely within the cartridge.

Description

[Technical field]

[0001] The present disclosure relates to the detection of infectious diseases, the identification of infectious microbial pathogens, and the determination of effective antimicrobial treatments for infectious diseases. This invention was made with Government support under Grant Nos. R43 AI080016, R01 AI117058, and R44 AI080016 awarded by the National Institutes of Health and Contract No. HHSO100201500022C awarded by the Biomedical Advanced Research and Development Authority. The Government has certain rights in the invention. [Background technology]

[0002] The epidemic of life-threatening infections caused by antibiotic-resistant bacteria is fueling a global healthcare crisis. Part of the problem is the fact that current diagnostic methods require several days to determine the optimal antimicrobial treatment to treat an infection. The delays caused by time-consuming tests result in suboptimal treatment, poor outcomes, and overuse of powerful broad-spectrum antibiotics that cause the spread of antibiotic resistance. Mortality due to infections caused by resistant bacteria is increasing exponentially. A 2014 report by the Review on Antimicrobial Resistance estimated that by 2050, antimicrobial resistance will be responsible for more than 10 million deaths per year.

[0003] Early treatment with optimally effective antibiotics targeted for the specific pathogen causing the infection can dramatically improve medical outcomes, including preventing death. Unfortunately, current methods for identifying effective targeted antibiotics, called antimicrobial susceptibility testing (AST) methods, require several days to deliver results. One reason that current antimicrobial susceptibility tests are so time-consuming is that the tests require large numbers of purified pathogen cells, on the order of millions. This requires a day or more using the more than 130-year-old colony purification method used to purify this number of cells by culturing in petri dishes. Once purified cells are available, it generally takes a day or more to identify the pathogen and to determine which antibiotics are effective to treat the patient.

[0004] Meanwhile, patients are "empirically" treated with broad-spectrum antibiotics that kill a wide range of pathogens that may cause infections. Although these drugs can treat a wide range of pathogens, they are generally not the optimal treatment for the patient's specific pathogen, but may not be able to effectively treat the infection completely. The empirical use of broad-spectrum antibiotics also leads to the spread of antibiotic resistance. These broad-acting drugs cause resistance not only to disease-causing pathogens, but also to trillions of harmless microorganisms that inhabit the human body. Further exacerbating the spread of antibiotic resistance is the fact that, in the absence of rapid diagnostics to determine which patients actually have an infection, non-infected patients are frequently unnecessarily treated with antibiotics that cause resistance.

[0005] Determining which antimicrobial treatments are effective as soon as possible using current methods can not only improve medical outcomes, but also lower the cost of health care. For example, common life-threatening hospital-acquired infections such as surgical site infections and ventilator-associated pneumonia are responsible for nearly $10 billion in health care costs in the United States. Hospital stays are the largest expense attributable to these infections. Placing patients on optimal antimicrobial therapy closer to the onset of symptoms can significantly accelerate their recovery and reduce long and costly hospitalizations.

[0006] In summary, current methods require several days to determine whether a patient has an infection and, if so, which antimicrobial agent is most likely to be effective. Primarily, this delay is due to the time-consuming cell purification steps that the methods require. In the absence of timely diagnostic information when a patient presents with symptoms, patients are empirically treated with broad-spectrum antibiotics, which may be suboptimal and may lead to the spread of resistance. Because antibiotics are prescribed before diagnostic results are available, even patients without infection are unnecessarily treated and acquire resistance. Summary of the Invention [Means for solving the problem]

[0007] The present invention addresses the need for a diagnostic test that detects infections and determines effective antimicrobial treatments much more rapidly than current methods. The present invention eliminates the time-consuming step required by today's methods to obtain large numbers of purified cells. The method of the present invention can detect infections and identify infectious agents and effective antimicrobial agents in hours rather than the days required by current methods. By detecting infections closer to the onset of symptoms and identifying effective targeted antimicrobial agents, the present invention has the potential to dramatically improve medical outcomes and minimize empirical treatment with broad-spectrum antibiotics that cause resistance.

[0008] Treating patients with effective antibiotics close to the onset of symptoms dramatically improves medical outcomes, saving both lives and medical costs. The systems and methods of the present invention allow for the delivery of antimicrobial susceptibility test results in hours, compared to the days required by current methods. Administering effective antibiotics earlier can improve medical outcomes and help reduce the overuse of empirically prescribed broad-spectrum antibiotics that cause the spread of resistance.

[0009] Traditional methods for determining effective treatments, called antimicrobial susceptibility testing, require several days to deliver results, in part because these methods require millions of purified pathogen cells. The process for obtaining these purified cells uses a time-consuming colony purification method for culturing disease-causing pathogen cells in a petri dish. Colony purification typically takes a day or more to complete. After colony purification, another day or more is generally required for antimicrobial susceptibility testing to identify the pathogen species and determine which antimicrobial should be used to treat the patient.

[0010] The present invention provides a method for identifying pathogens or determining antimicrobial susceptibility from a specimen without colony or cell purification. The significant improvement in time to result for the methods of the present invention compared to current methods comes from the ability of the present invention to directly analyze patient specimens without the need for the time-consuming step of preparing large numbers of purified pathogen cells.

[0011] Antimicrobial susceptibility testing may be considered a step-by-step process. The objective is to determine which members of a panel of antimicrobial agents are effective against the particular pathogen strain causing the patient's infection. Typically, when an infection is detected, the species of the pathogen is first identified. Identifying the species of the pathogen is useful for selecting antimicrobial agents and medications that can be commonly used to treat that species. However, the particular pathogen strain causing the infection may have become resistant to any of the antimicrobial agents, so antimicrobial susceptibility testing must be performed to determine the potential treatments to which the pathogen is actually susceptible.

[0012] After species identification, pathogen cells from the patient specimen are apportioned, or aliquoted, into a series of liquid solutions including nutrient growth media containing various antimicrobials at different concentrations. The aliquots are then incubated at a temperature conducive to microbial replication (typically 35-37°C). If the pathogen is susceptible to an antimicrobial, it can replicate normally, i.e., the number of pathogen cells will increase as in microbiological growth media in the absence of the antimicrobial. If the pathogen is susceptible to an antimicrobial, it will not replicate, will replicate to a much lower extent, or will exhibit morphological or other abnormalities, which indicate the effectiveness of the antimicrobial. Finally, replication of pathogen cells is evaluated in the various aliquots to determine which antimicrobials are effective. We refer to the set of antimicrobial susceptibility / resistance results of a pathogen for a series of antimicrobials as its antimicrobial susceptibility profile.

[0013] Both the conventional method and the method of the present invention for antimicrobial susceptibility testing follow the steps described above, but the method of the present invention determines the antimicrobial susceptibility profile of a pathogen in a few hours, whereas the conventional method requires several days. The rapid antimicrobial susceptibility test results using the method of the present invention arise from the ability of the new method to directly test patient specimens without time-consuming culture-based pre-enrichment growth to achieve high concentrations of pure cells. This enrichment and purification is most commonly performed using colony purification on petri dishes.

[0014] For conventional methods, cells recovered after colony purification are first identified using biochemical, microbiological, nucleic acid methods or matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). Once the identity of the pathogen species is known, appropriate antimicrobials and concentrations can be selected that are appropriate for determining the antimicrobial susceptibility profile for that species of pathogen.

[0015] Several novel aspects of the systems and methods of the present invention enable the ability to rapidly deliver antimicrobial susceptibility results directly from patient specimens.

[0016] First, patient specimens generally contain several orders of magnitude fewer cells than are required for traditional antimicrobial susceptibility testing. In contrast to current culture-preconcentration dependent methods, the method of the present invention can enumerate small numbers of pathogen cells by sensitive single cell counting using non-magnified digital imaging. Furthermore, because the method enumerates small numbers of individual cells, it can very quickly determine, with only a few generations of bacteria, whether cells are increased in number in aliquots containing antimicrobials and growth medium.

[0017] Second, patient specimens contain sample matrix and commensal microorganisms that are not associated with infectious pathogens. Guidelines for conventional methods, such as those from the Clinical Laboratories Standards Institute (CLSI) or the European Committee on Antimicrobial Susceptibility Testing (ECAST), require purified culture cells resulting from clonal growth of colonies on an agar-based growth medium in a Petri dish. These cells contain only a single microbial species and no sample matrix.

[0018] As discussed above, the identity of the pathogen species must be known in order to accurately interpret antimicrobial susceptibility test results to arrive at effective clinical treatment options, which is an important reason underlying why most conventional and emerging antimicrobial susceptibility testing methods require pure cultures of cells.

[0019] To determine antimicrobial susceptibility profiles as described above, most conventional and emerging methods evaluate the effect of different concentrations of different antimicrobials on the growth of the target pathogen. The reason these methods require a pure population of identified cells to interpret antimicrobial susceptibility test results is because they use non-specific methods, e.g., light scattering or microscopy, to evaluate growth in the antimicrobial-containing aliquots. Consider the case when there are more than one species, e.g., a pathogen and a species of normal microorganisms that are part of the human microbiome - which is the case in most primary patient specimens. If growth is observed in the antimicrobial-containing aliquot, it is not possible to say using a general method to detect growth whether the disease-causing pathogen, or one or more of the commensal species, were resistant and capable of growing.

[0020] In contrast to conventional methods and other methods that require purified pathogen cells, which use non-specific methods to detect whether a pathogen grows in the presence of an antimicrobial drug, the method of the present invention uses pathogen-specific detection to evaluate the growth of a pathogen in the presence of various antimicrobial drugs. Because only disease-causing pathogen cells are enumerated after the incubation step (and not any commensal microorganisms), the method of the present invention can be used to determine antimicrobial susceptibility directly in non-sterile primary specimens containing one or many commensal microbial species.

[0021] The present systems and methods for pathogen identification can be used to determine whether a specimen contains cells of a pathogen species in sufficient numbers to be suspected of causing an infectious disease.

[0022] The present systems and methods for antimicrobial susceptibility testing can be used to determine which of one or more antimicrobial agents are capable of preventing normal cellular replication of a pathogen suspected of causing an infection in a patient specimen. Such antimicrobial agents can potentially be used to effectively treat the infection in the patient.

[0023] In a preferred embodiment of the method of the present invention for antimicrobial susceptibility testing, the specimen is divided into portions containing a nutrient growth medium to promote microbiological cell replication or growth. One or more of said portions may be used as a reference or baseline portion that is directly processed and analyzed by the method of the present invention prior to incubation at a temperature that promotes growth to determine the number and amount of pathogen cells.

[0024] One or more of these portions may be incubated at a temperature that promotes pathogen cell growth to determine whether the pathogen cells are viable, and other portions each contain a specific concentration of one or more antimicrobial agents and are incubated to determine the effect of the antimicrobial agents on pathogen cell replication.

[0025] The system and method of the present invention are then used to analyze the number and amount of pathogen cells in the incubated portion and compare these results to the number and amount of pathogen cells in a non-incubated reference portion. If the pathogen cells are significantly impaired in their ability to replicate normally in the portion containing a particular antimicrobial agent, the pathogen is scored by the analysis software as being susceptible to the antimicrobial agent at the concentration in that portion. Alternatively, if normal growth is not impaired in the portion, the pathogen is scored by the analysis software as not susceptible to the antimicrobial agent at the concentration in that portion.

[0026] The systems and methods of the present invention include criteria by which cellular replication is evaluated for determination of antimicrobial susceptibility or resistance of pathogens or microbial targets in the antimicrobial-containing portion. These criteria may be determined for a particular combination of parameters, including but not limited to target microbial species, specimen type, antimicrobial agent, growth medium composition, temperature, and incubation time.

[0027] The evaluation criteria are preferably empirically determined by correlation with a recognized reference method for antimicrobial susceptibility testing. One standard reference method is the broth microdilution method (BMD), a method that has been well described elsewhere (see Patel, 2015, M07-A10, CLSI 35(2), incorporated by reference) and is understood by those skilled in the art. Broth microdilution is a method in which the antimicrobial susceptibility of a microbial strain to an antimicrobial agent is evaluated under standard conditions. Purified cells of the target microorganism are added at a defined concentration to a series of portions or aliquots of a defined nutrient growth medium containing two-fold serial dilutions of various antimicrobial agents. The antimicrobial susceptibility of the microbial strain is determined after visually assessing the turbidity of the various portions after a defined period of growth incubation. The lowest concentration of antimicrobial agent at which turbidity is visually absent or significantly reduced compared to the portion without antimicrobial agent is called the minimum inhibitory concentration (MIC). Organizations that determine standards for antimicrobial susceptibility testing (e.g., CLSI or EUCAST) correlate MIC values ​​for a combination of a particular microbial species and a particular antimicrobial agent with the effectiveness of a particular therapeutic dose of the antimicrobial agent in clinical practice. MIC values ​​are commonly binned into categorical ranges: susceptible, intermediate resistance, and resistance. These are called SIR or categorical antimicrobial susceptibility test results. In this way, the MIC for a particular strain of a particular species against a particular antimicrobial agent can be reported as a SIR or categorical result. Other standard methods used to determine include -Bauer or disk diffusion and agar dilution. These methods are described in CLSI and EUCAST documents and are known to those skilled in the art.

[0028] The evaluation criteria for determining antimicrobial susceptibility test results using the method and system of the present invention are empirically determined by using the present invention to evaluate the degree and quality of cell replication of various strains of a particular species at various concentrations of a particular antimicrobial agent, similar to broth microdilution. The criteria for assigning antimicrobial susceptibility test results (generally MIC or SIR categorical results) to strains of a particular species for a particular antimicrobial agent are selected so that the results for various strains are consistently correlated with the results of the reference broth microdilution method using the present invention. For example, a criterion that can be used by the system and method of the present invention for determining antimicrobial susceptibility test results is the evaluation of the growth fold (the fold increase in the number of target cells) of a target microorganism of a particular species over a certain period of incubation in a nutrient growth medium at a certain temperature in the presence of various concentrations of a particular antimicrobial agent. In this case, the empirical study to determine the effective criteria for use with the system and method of the present invention for evaluating antimicrobial susceptibility evaluates the growth fold of various strains of a species at various concentrations of an antimicrobial agent. A threshold for growth fold can be selected such that the growth fold measured for a strain using the present invention correlates with the broth microdilution results for the same strain grown in the presence of the same antimicrobial agent. The threshold is selected empirically using various strains of the target species such that if the growth fold of a strain exceeds the threshold, the strain is categorized by the present invention as having significant growth in the presence of that concentration of antimicrobial agent. If the growth fold of a strain is below the growth fold threshold, the strain is categorized by the present invention as not having significant growth. Thus, in this example, the threshold for growth fold is selected such that both the reference broth microdilution method and the method of the present invention return the same results as to whether various strains are determined to have significant growth or not at various antimicrobial agent concentrations.

[0029] Other evaluation criteria can also be used by the system and method of the present invention to determine the results of antimicrobial susceptibility testing. For example, evaluation of morphological characteristics that reflect the disruption of normal cell replication caused by incubation in an antimicrobial agent. As another example, the degree of growth inhibition in the portion containing the antimicrobial agent compared to the portion not containing the antibiotic after the incubation step can be evaluated. Multiple evaluation criteria can also be used simultaneously to determine whether an antimicrobial agent causes significant disruption of normal cell replication of target cells at a particular concentration.

[0030] Thus, the methods of the present invention can detect infectious diseases directly from patient specimens, identify infectious pathogens, and determine which antimicrobial agents are effective for treatment.

[0031] A patient specimen, e.g., urine, feces, respiratory, wound, cerebrospinal fluid or blood, is preferably transferred directly into the analysis cartridge for microbial analysis without any specimen preparation by the user. Thus, there is preferably no need for the user to perform colony purification, nucleic acid purification, or other time- or labor-intensive specimen preparation protocols to isolate a large number of pure pathogen cells. The specimen is preferably loaded directly into the test cartridge without prior enrichment or purification, e.g., to remove biological debris. The cartridge contains the reagents for microbial quantification and identification and antimicrobial susceptibility testing of the present disclosure. All steps, such as species-specific labeling and imaging of microorganisms in the specimen, are performed on the cartridge on which the specimen is directly loaded. The cartridge contains target cell-specific labels, e.g., fluorescent probes, that are used to identify microorganisms in the specimen by the systems and methods of the present invention. To identify microorganisms, the analyzer preferably includes an imaging subsystem for imaging the labeled microorganisms in the cartridge.

[0032] Rapidly detecting infectious diseases, identifying pathogens, and determining antimicrobial susceptibility using the systems and methods of the present invention offers the potential to deliver actionable results to guide effective treatment of patient infections much more quickly than traditional methods that require lengthy culture steps. The systems and methods of the present invention can provide clinicians with the ability to detect infectious diseases and identify infectious pathogens in about 30 minutes, and determine antimicrobial susceptibility test results in a few hours, simply by transferring a patient specimen directly into an analytical cartridge and loading the cartridge into an instrument.

[0033] Embodiments of the present invention include the detection of microorganisms directly from patient specimens and the identification of effective treatments.

[0034] In certain aspects, the disclosure provides a method for antimicrobial susceptibility testing. The method includes obtaining a polymicrobial specimen; dividing the specimen into wells, at least some of the wells containing one or more agents at different drugs or concentrations; incubating the specimen in the wells to allow differential growth in response to the different drugs or concentrations; and counting individual cells of a particular species in each well to identify an agent or concentration of an agent that inhibits growth of the particular species. The method may include counting cells in a control well that does not contain an antimicrobial agent (e.g., after incubation of the control wells at a defined temperature of 30-40° C. for a defined time of 1-12 hours). The method may include comparing the number of cells counted in each well with the count from the control well to determine the survival rate of the microorganism when exposed to the different drugs or concentrations of the drug. The incubating step may include fluorescently labeling the microorganism in a species-specific manner, and the counting step may include imaging each well and counting fluorescent spots in the image. The labeling step may use target-specific fluorophore-labeled nucleic acid or nucleic acid analog probes, and the counting step may utilize fluorescent in situ hybridization. Preferably, the steps do not include nucleic acid amplification. The incubating step may last less than about 1 hour and may be performed in growth medium at a temperature below about 40°C.

[0035] In certain embodiments, the splitting, incubating and counting steps are performed using a cartridge containing wells. The method may include transferring a portion of the specimen into the cartridge and loading the cartridge into an analyzer. The cartridge may include microorganism-binding magnetic beads, and the analyzer may use a magnet to separate the microorganisms from other portions of the specimen and perform the counting step using an imaging subsystem. In some embodiments, the analyzer uses a pneumatic subsystem to perform the splitting step in a cartridge in which the wells are in the cartridge and are preloaded with different drugs or different concentrations of drugs, and after the splitting and incubation steps, the analyzer transfers the contents of the wells into the corresponding reagent wells to expose the incubated specimen therein to the magnetically-bound magnetic beads and the species-specific detectable labels. The analyzer may use a magnet to attract the microorganism-binding magnetic beads and the bound individual microorganisms to a detection surface in the cartridge.

[0036] In some embodiments, the magnet attracts the microorganism-bound magnetic beads through a dye cushion that displaces unbound detectable label from the detection surface. The analyzer may use a carousel and / or a mechanical cartridge transporter to move the cartridge to an imaging subsystem where the counting step may be performed. In some embodiments, the species-specific detectable label comprises a fluorescent nucleic acid probe that hybridizes to target nucleic acid of a particular species of microorganism. The incubating and counting steps may achieve substantially fluorescent in situ hybridization (FISH) analysis at physiological temperatures within the analyzer.

[0037] Identifying antimicrobial agents that inhibit microbial growth may include comparing the number of complexes in the incubated sample with the number of complexes in an unincubated sample. The analyzer may use the cartridge to perform species-specific enumeration of individual microorganisms after differential growth.

[0038] In certain embodiments, the well is in a cartridge and the method includes transferring a portion of the specimen into the cartridge and loading the cartridge into an analyzer. The analyzer manipulates the cartridge to perform the splitting, incubating, and counting steps. The specimen does not require any chemical or molecular sample preparation techniques by the user prior to the transferring step. The transferring step may be performed by pipetting a portion of the specimen from a collection container into a sample well on the cartridge.

[0039] In some aspects, the present disclosure provides a method for analyzing microorganisms. The method includes the steps of obtaining a specimen containing microorganisms; transferring a portion of the specimen into a well without any colony or cell purification or culture step, the well containing a species-specific detectable label and a microorganism-binding magnetic bead; using a magnet to collect the beads on an imaging surface; and imaging the detectable label on the imaging surface to thereby determine the presence of the species of cells in the portion of the specimen. The specimen does not require any chemical or molecular sample preparation technique by the user before the transferring step. The method may further include the steps of labeling the target microorganism with a species-specific detectable label in the well, and binding the microorganism to the microorganism-binding magnetic bead, and the observing step includes imaging the labeled magnet-bound cells without washing away the unbound detectable label. The method may further include the step of counting the individual cells of the labeled magnet-bound cells.

[0040] In certain embodiments, the wells are provided in the cartridge and the steps of collecting and imaging occur while the beads and cells are in the cartridge.

[0041] Loading the cartridge into an analyzer that performs the collecting and imaging steps. The analyzer may include multiple subsystems, including at least one imaging subsystem for imaging the labeled microorganisms, and a carousel operable to transport the test cartridges between the subsystems.

[0042] The transferring step may include transferring the specimen into a receiving well of the cartridge, after which the analyzer may split the specimen into a number of split wells in the cartridge (the well being one of the split wells). The species-specific detectable label may include a fluorescently labeled oligonucleotide probe that specifically hybridizes with nucleic acid of cells of the species. The imaging step may include fluorescent in situ hybridization (FISH) analysis at a constant physiological temperature within the cartridge.

[0043] In some embodiments, collecting the beads on the imaging surface comprises magnetically attracting the beads through a dye cushion in the well while the dye cushion displaces unbound detectable label from the imaging surface. Preferably, the cells of the species are determined to be present in the specimen within about 30 minutes of the transferring step.

[0044] In some embodiments, the invention includes microbial detection and identification of effective treatments directly from patient specimens. In certain aspects, the invention provides for identification of microorganisms. Microorganism identification is achieved directly from patient specimens, such as whole blood, plasma, serum, urine, sputum, saliva, feces, cerebrospinal fluid, amniotic fluid, peritoneal fluid, pus, lymph, vaginal secretions, nasal secretions, vomit, sweat, and tissue, without a specimen preparation step. For example, the method may include transferring a urine specimen directly into an analytical cartridge device for microbial identification. In some embodiments, the method includes transferring a patient specimen directly into a sample well of an analytical cartridge, and manipulating the cartridge to label the microorganisms in a species-specific manner, image the labeled microorganisms, and identify the labeled microorganisms. Labeling the microorganisms includes exposing the specimen to a species-specific label. The label may include a target-specific magnetic tag and a detectable label. The detectable label may include a species-specific fluorescent probe. The probe may preferentially only detect the nucleic acid of the species of the microorganism. When exposed to a particular species of microorganism, the probe hybridizes only to that species allowing for detection of that species of microorganism. In this way, multiple species of microorganisms in a sample can be identified and tested for susceptibility to different antimicrobials or other treatments in a single analytical cartridge. In some embodiments, operating the cartridge may include loading the cartridge into an instrument to identify microorganisms in a patient sample in the cartridge. The present invention provides analytical cartridges and instruments that can directly accept and process patient samples to identify microorganisms and / or determine therapeutic susceptibility and efficacy all within the cartridge.

[0045] The systems and methods of the present invention include instruments or analyzers that can be used to interact with the analytical cartridge to perform the methods of the present invention. The instruments can include multiple subsystems for performing the methods of the present invention. The analytical cartridge can be loaded into an instrument that has multiple subsystems for processing the specimen in the cartridge. In a preferred embodiment, one of the multiple subsystems can be an imaging subsystem for imaging the labeled microorganisms in the cartridge. The instrument subsystems can also include a pneumatic subsystem, a magnetic subsystem, and a waste subsystem. The instrument can also include a carousel, a mechanical cartridge carrier, and a task scheduler for moving and manipulating the cartridge within the instrument. The instrument can perform all of the processing steps at a constant temperature.

[0046] The systems and methods of the present invention, including instruments and devices, can perform a wide range of diagnostic functions, including detecting infection; detecting, identifying and quantifying all types of pathogen cells; determining antimicrobial susceptibility; detecting and quantifying biomarkers, including toxins, viruses, and host-response factors; and detecting and quantifying diagnostically informative human or host cells. The systems and methods of the present invention are capable of performing such diagnostic functions, alone or in combination, simultaneously on a single specimen in a single device (e.g., analytical cartridge). The systems and methods of the present invention include the capability of random access processing, such that multiple such devices performing different types of diagnostic tests (e.g., for urinary tract infections, blood infections, and respiratory infections) can be processed simultaneously on a single instrument. Thus, the instruments and cartridges described herein provide such functionality and can be manipulated accordingly to process specimens within the device.

[0047] The methods and systems of the present invention can include transporting analytical cartridges via a carousel within the instrument to multiple subsystems of the instrument. In some embodiments of the present invention, a mechanical transport mechanism within the instrument moves each analytical cartridge between the carousel and the various subsystems of the instrument. The mechanical cartridge transporter applies a pushing force to move the cartridge to and from the carousel. In some embodiments, the instrument includes a task scheduler for managing the transportation and movement of each analytical cartridge among the multiple subsystems.

[0048] In some embodiments, management of the movement of each analytical cartridge within the instrument, including the time each cartridge spends in the subsystems, may be performed by a task scheduler within the instrument. The method may include reserving the time in the various subsystems required for each cartridge. The method may also include operating a mechanical cartridge carrier that moves the cartridge from one of the subsystems to the carousel and rotates the carousel to a position for the cartridge adjacent one of the other subsystems. In some embodiments of the invention, the task scheduler may manage the movement of the analytical cartridge (i.e., the steps / parameters of the analysis to be performed) by identifying the contents of the cartridge. The contents of the cartridge and the required processing may be associated with a tag on the cartridge. In some embodiments of the invention, each analytical cartridge includes a tag readable by the instrument. The instrument may read the tag via a barcode analyzer and associate the tag with a particular set of instructions that the task scheduler executes. The tag may be a barcode and may be specific / unique to the analyte in the instrument.

[0049] The systems and methods of the invention include pneumatically dispensing the analyte within the cartridge. In some embodiments, the method includes dispensing the analyte from the sample well to multiple wells within the cartridge. The method further includes controlling the distribution of the analyte by operation of multiple valves within the cartridge. In certain embodiments of the invention, the method includes transporting the cartridge to a pneumatic subsystem of the instrument and operating the subsystem to dispense the analyte from the sample well to multiple wells within the cartridge.

[0050] The system and method of the present invention are used to label microorganisms. The method of labeling microorganisms includes loading the specimen directly into an analytical cartridge containing specific reagents for processing the specimen according to the method of the present invention. The reagents may include, for example, species-specific labels, growth media, antimicrobial agents, and dyes. The reagents may be in liquid form, or preferably lyophilized, and may be loaded into certain wells in the analytical cartridge (e.g., test device). The reagents may be specific to the test, for example, the cartridge may be test specific. The method of the present invention includes labeling microorganisms by exposing the specimen to species-specific magnetic tags and detectable labels preloaded in the cartridge. The specimen may be exposed to the magnetic tags and detectable labels in the wells of the cartridge by pneumatic dispensing. The detectable labels may include fluorescent oligonucleotide or antibody probes, specific and non-specific ligands, lectins, dyes, and dyes that bind to targets. In certain embodiments, the magnetic tags are used in combination with detectable labels that bind to the target microorganisms before magnetically selecting and imaging the target microorganisms. In some embodiments, the detectable label may be a fluorescent probe that specifically hybridizes with the nucleic acid of a species of microorganism. In a preferred embodiment, the detectable label is a fluorescent rRNA-targeted fluorescent oligonucleotide probe that hybridizes with a specific species of microorganism. In certain embodiments, the labeling and imaging method includes fluorescent in situ hybridization (FISH) to label the cellular targets, and magnetic selection of the labeled cellular targets, which places the labeled targets on a detection surface for imaging and analysis. In certain embodiments, the labeling and magnetic selection steps occur at a constant temperature. In some preferred embodiments, these steps are all performed at a temperature of about 36-39°C.

[0051] In certain embodiments of the invention, the method includes transporting the cartridge to a magnetic subsystem of the instrument and operating the magnetic subsystem to draw the labeled microorganisms through an opaque, dense, aqueous dye cushion layer to a detection surface within the cartridge. The dye cushion layer can be designed to optically isolate unbound fluorescent label and analyte matrix from the detection surface. This can eliminate the need for a washing step to remove fluorescent label that is not bound to the target cells, increase the signal to noise, and minimize or eliminate the need for a sample preparation step. The method includes drawing the labeled microorganisms through the dye cushion layer before they reach the detection surface.

[0052] The systems and methods of the present invention provide imaging analysis of specimens. The specimens are directly processed and imaged in the cartridge to determine the identity of the microorganisms present in the specimen. In particular, the detection surface of the cartridge is imaged and the imaging analysis identifies the microorganisms in the specimen. The method of identifying the microorganisms may also include quantifying the microorganisms. In certain embodiments of the present invention, the method includes transporting the cartridge to an imaging subsystem of the instrument by operating a carousel, and operating the subsystem to image and analyze the detection surface of the cartridge. Digital, non-magnifying techniques can be used to identify and quantify the microorganisms.

[0053] The specimen may also be incubated in the presence of antimicrobial agents or other treatments of various kinds at various concentrations prior to imaging. The incubation may occur in the same analytical cartridge or in a different analytical cartridge. Regardless, the specimen processed as described herein may be processed at a constant temperature, including the incubation steps described herein. The constant temperature may be the physiological temperature of the mammal. The physiological temperature may be a temperature below 40°C. In a preferred embodiment, the temperature is between 36 and 39°C.

[0054] The system and method of the present invention can be used to identify microorganisms and test for antimicrobial susceptibility of microorganisms in a specimen. The method of the present invention may be performed in a separate analysis cartridge or in the same cartridge. In a preferred embodiment of the present invention, the analysis cartridge directly receives the specimen and divides the specimen into separate portions internally, where one or more portions may be exposed to one or more species-specific detectable labels. The portions are processed and imaged to identify the microorganisms in each portion. In certain embodiments, identification and antimicrobial susceptibility testing can be performed on one and the same specimen in the same device.

[0055] The method of the present invention includes antimicrobial susceptibility testing of a multi-microbial specimen. The method includes distributing a multi-microbial specimen from a subject into multiple compartments. One or more compartments may contain different antimicrobial agents and may be incubated in the presence of different antimicrobial agents to determine differential growth in the incubated portions. One or more compartments may not be exposed to antimicrobial agents to provide a baseline for determining changes in other compartments. The compartments may also be incubated in the presence of growth medium. The compartments are exposed to species-specific detectable labels. The compartments are imaged to identify and quantify the microorganisms in each compartment. Analyzing the number of microorganisms in each compartment identifies the effectiveness of each antimicrobial agent in reducing or even preventing the growth of pathogenic agents. The effectiveness of treatment is determined by counting and comparing the number of microorganisms in each compartment. In some embodiments, the method includes comparing the number of microorganisms in the incubated compartment to the number of microorganisms in the baseline compartment. In certain aspects, the method of antimicrobial susceptibility testing may be performed by placing the specimen directly into an analytical cartridge. The analytical cartridge divides the specimen into multiple compartments within the cartridge for antimicrobial susceptibility testing. The method of antimicrobial susceptibility testing is performed within the time of introducing the cartridge into the instrument, preferably within about 4 hours.

[0056] In certain aspects, the present invention provides a system for identifying and determining antimicrobial susceptibility of microorganisms in a specimen. A preferred system includes an analytical cartridge and is operable to receive a patient specimen and includes an instrument operable to receive the analytical cartridge. In certain embodiments, an analytical cartridge for identification, antimicrobial susceptibility testing is provided preloaded with reagents such as species-specific labels, antimicrobial agents, and growth media specific for various microorganisms. In some embodiments, the analytical cartridge contains multiple wells (or compartments) within the cartridge. The multiple wells within the cartridge may include at least a sample well, a distribution well, and an imaging well. In some embodiments, the system of the present invention distributes the specimen from the sample well to multiple wells within the cartridge. In certain aspects, the specimen is distributed from the sample well to multiple distribution wells preloaded with antimicrobial agents and growth media for incubation. The incubated specimen may then be transferred from the distribution well to a reagent well that exposes the incubated specimen to a species-specific label to form a complex.

[0057] A user may select the appropriate application-specific cartridge and directly transfer patient samples therein to perform specific diagnostic tests and analyses. The cartridge may be loaded into an instrument that processes the sample with the applicable analysis. The instrument may identify the required processing steps by scanning a tag, such as a barcode, on the cartridge, where the tag is specific to the analysis to be performed.

[0058] Results obtained using the systems and methods of the invention may be used to inform treatment decisions. The methods may include administering a treatment to a patient identified as being effective in reducing the growth or survival rate of a microorganism identified as a pathogenic agent. [Brief description of the drawings]

[0059] [Figure 1]FIG. 1 is a schematic diagram of the method for antimicrobial susceptibility testing.

[0060] [Diagram 2] FIG. 2 shows the cartridge.

[0061] [Diagram 3] FIG. 3 is a schematic diagram of steps of another exemplary method of the present invention.

[0062] [Figure 4] FIG. 4 shows the device.

[0063] [Diagram 5] FIG. 5 is a top view of the components within the device.

[0064] [Figure 6] Figure 6 is an example of a workflow for performing antimicrobial susceptibility testing.

[0065] [Figure 7] FIG. 7 shows the transfer of specimens into the sample wells of the cartridge.

[0066] [Figure 8] FIG. 8 shows the cartridges being loaded into the loading tray.

[0067] [Figure 9] FIG. 9 shows the number of individual E. coli cells counted.

[0068] [Figure 10] FIG. 10 shows the number of Enterococcus faecalis cells counted.

[0069] [Figure 11] FIG. 11 gives the results of the test.

[0070] [Figure 12]FIG. 12 shows the results of another test.

[0071] [Figure 13] FIG. 13 shows the growth results.

[0072] [Figure 14] Figure 14 shows that the limit of detection (LoD) for E. coli ATCC 19138 is shown. The upper limit of blank (LoB) was 89 CFU per assay and the LoD was 284 CFU per assay. This corresponds to a LoD of 9,467 CFU per ml of urine.

[0073] [Figure 15] Figure 15 shows that the limit of detection (LoD) for P. aeruginosa ATCC 9721 is shown. The upper limit of blank (LoB) was 104 CFU per assay and the LoD was 506 CFU per assay. This corresponds to a LoD of 16,867 CFU per ml of urine.

[0074] [Figure 16] Figure 16 shows that the limit of detection (LoD) for K. pneumoniae ATCC 700603 is shown. The upper limit of blank (LoB) was 109 CFU per assay and the LoD was 319 CFU per assay. This corresponds to a LoD of 10,633 CFU per ml of urine.

[0075] [Figure 17] FIG. 17 is a table of the probe sequences used in this example.

[0076] [Figure 18] Figure 18 shows that the average signal (n=3) is plotted for 11 E. coli strains for input cell concentrations of approximately 600 CFU per assay (light grey bars) and 3000 CFU per assay (dark grey bars). The signal from a no-cell control (blank) is shown at the far left of the figure. Error bars represent 1 standard deviation.

[0077] [Figure 19] Figure 19 shows the percentage of input cells detected (determined by plate counting) is shown for each of the 11 E. coli strains. Each bar represents the average of 6 determinations in triplicate from each of the two different input cell levels. Percentage of cells detected was calculated as [(assay signal-background signal) / input cells] x 100.

[0078] [Figure 20] FIG. 20 is a table giving the inclusion results for four additional bacteria.

[0079] [Figure 21] FIG. 21 is a table giving the probe sequences used in this example.

[0080] [Figure 22] FIG. 22 is a table showing challenge bacteria for testing specificity for detecting E. coli.

[0081] [Diagram 23] FIG. 23 is a table showing the probe sequences used in this example.

[0082] [Figure 24] FIG. 24 shows the specific detection of E. coli and non-detection of the eight challenge bacteria.

[0083] [Diagram 25] FIG. 25 shows the specific detection of E. coli and non-detection of eight additional challenge bacteria.

[0084] [Figure 26] Figure 26 is an image showing the same field of view captured in four different color channels using CCD imaging and four different fluorophores, one for each bacterium. All four bacteria could be detected in a single well.

[0085] [Figure 27] FIG. 27 is a table of the probe sequences used in this Example 4.

[0086] [Figure 28] FIG. 28 shows BIUR0017 with nitrofurantoin.

[0087] [Figure 29] FIG. 29 shows BIUR047 with cefazolin.

[0088] [Diagram 30] FIG. 30 shows BIUR057 with ciprofloxacin.

[0089] [Diagram 31] FIG. 31 shows BIUR052 with trimethoprim / sulfamethoxazole.

[0090] [Diagram 32] FIG. 32 is a table of the probe sequences used in this Example 6.

[0091] [Diagram 33] FIG. 33 is a visual comparison of normal bacteria (left panel) to filamentous fungi (right panel).

[0092] [Diagram 34] FIG. 34 shows that the MIC obtained by the novel rapid AST method described in this invention is called at 0.25 μg / mL.

[0093] [Diagram 35] FIG. 35 is a table of AST results for all bacteria and antibiotics tested in this example.

[0094] [Diagram 36]FIG. 36 is a table of the probe sequences used in this Example 7.

[0095] [Figure 37] FIG. 37 shows the Multipath™ UTI-AST cartridge.

[0096] [Figure 38] FIG. 38 is a table showing the antibiotic concentrations tested.

[0097] [Figure 39] FIG. 39 is a table of the oligonucleotides used in this Example 8.

[0098] [Diagram 40] Figure 40 shows the results for BIUR0067.

[0099] [Diagram 41] Figure 41 shows the results for BIUR0084.

[0100] [Diagram 42] FIG. 42 is a summary of the overall intrinsic and categorical agreement for all organisms, antibiotics and inoculum levels.

[0101] [Diagram 43] FIG. 43 shows the MIC results for various inoculum levels obtained using the new method described herein compared to the conventional BMD method.

[0102] [Diagram 44] FIG. 44 summarizes the MIC results obtained for various inoculum levels.

[0103] [Diagram 45] FIG. 45 is a table of the probe sequences used in this Example 9.

[0104] [Figure 46]FIG. 46 shows that the E. coli MICs are consistent with the above method for various inoculations of S. aureus, while the BMD MICs increase with increasing S. aureus.

[0105] [Figure 47] FIG. 47 shows a summary of the concordance for E. coli with off-target microorganisms at various inoculum levels against standard BMD.

[0106] [Figure 48] FIG. 48 shows the concordance of E. coli with various inoculum levels of standard BMD of off-target microorganisms (S. aureus, Staphylococcus epidermidis, and Citrobacter freundii).

[0107] [Figure 49] FIG. 49 shows the concordance of E. coli with various inoculum levels of standard BMD of off-target microorganisms (Micrococcus luteus, Acinetobacter baumannii, Corynebacterium minutissimum).

[0108] [Figure 50] FIG. 50 shows the concordance of E. coli with various inoculum levels of the standard BMD of an off-target microorganism (K. pneumoniae).

[0109] [Figure 51] FIG. 51 is a table of the probe sequences used in this Example 10.

[0110] [Figure 52] FIG. 52 is a comparison of the new rapid AST method and the BMD method for determining the MIC of imipenem for E. coli in the presence of carbapenem-hydrolyzing B-lactamase resistant strains of K. pneumoniae.

[0111] [Figure 53] FIG. 53 shows that the E. coli MICs are consistent with the above method for various inoculations of a carbapenem-hydrolyzing B-lactamase resistant strain of K. pneumoniae, while the standard BMDs are not.

[0112] [Figure 54] FIG. 54 is a table of the probe sequences used in this Example 11.

[0113] [Figure 55] FIG. 55 shows the essential agreement across 15 urines.

[0114] [Figure 56] Figure 56 shows 100% essential agreement and 100% categorical agreement for each of the 15 spiked culture-negative clinical UTI urine samples against standard BMD. *Cefazolin, ciprofloxacin, levofloxacin, nitrofurantoin and trimethoprim-sulfamethoxazole.

[0115] [Figure 57] Figure 57 shows the MICs of 15 urine samples spiked with E. coli determined by the new AST method compared to the standard BMD method ("CLSI compliant"). Concentration (μg / ml).

[0116] [Figure 58] FIG. 58 is a table of the probe sequences used in this Example 12.

[0117] [Figure 59] FIG. 59 shows the ciprofloxacin-susceptible and -resistant strains used in this example.

[0118] [Figure 60] FIG. 60 is the first half of a table of the probe sequences used in this Example 13.

[0119] [Figure 61] FIG. 61 is the second half of a table of the probe sequences used in this Example 13.

[0120] [Figure 62] Figure 62 shows the essential agreement for polymicrobial infections with two target organisms. As can be seen below, the AST method gives an essential agreement of 100% with the standard BMD.

[0121] [Figure 63] Figure 63 shows the category agreement for polymicrobial infections with two target organisms. As can be seen below, the AST method provides 100% category agreement with standard BMD.

[0122] [Figure 64] FIG. 64 shows that the target pathogens are detected only in wells containing their species-specific DNA oligonucleotide FISH probes.

[0123] [Figure 65] FIG. 65 shows that the target pathogens are detected only in wells containing their species-specific DNA oligonucleotide FISH probes.

[0124] [Figure 66] FIG. 66 shows that the target pathogens are detected only in wells containing their species-specific DNA oligonucleotide FISH probes.

[0125] [Figure 67] FIG. 67 is a table, "Table A of Example 14," which shows that the target pathogens were detected while other non-target pathogens were not detected.

[0126] [Figure 68] FIG. 68 is a table, “Table B of Example 14,” showing the probe sequences used in this Example 14. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0127] The present invention provides a system and method for rapid, automated analysis of microorganisms without specimen preparation. The system and method of the present invention provides microbial identification and antimicrobial susceptibility testing (AST) directly from a patient specimen. Microorganisms in a specimen can be analyzed for differential growth in the presence of various antimicrobial agents. Specimen manipulation, incubation, processing and analysis steps are all performed within a single analysis cartridge. Identification analysis can be completed in about 30 minutes and antimicrobial susceptibility testing can be completed in about 4 1 / 2 hours. The system and method of the present invention uses species-specific labeling, and non-magnified digital imaging and image analysis to identify targets such as microorganisms to accurately and rapidly identify and quantify targets in a specimen. The system and method of the present invention enables microbial identification and antimicrobial susceptibility testing analysis in specimens that have preferably not been processed to purify target pathogen cells and that preferably do not require manual specimen preparation by the user. With minimal need for user interaction with the specimen, the system and method of the present invention provides microbial identification and antimicrobial susceptibility testing analysis results directly from a patient specimen within minutes and hours, respectively, compared to the days required in the prior art. The analytical cartridge can be automatically processed by the instrument to perform each of the steps necessary to identify the microorganism and to perform the antimicrobial susceptibility testing analysis.

[0128] FIG. 1 is a schematic diagram of a method 101 for antimicrobial susceptibility testing. The method 101 includes a step 105 of obtaining a polymicrobial specimen. Without any sample preparation, colony purification or other methods, the raw original specimen is divided into wells 109. At least some of the wells contain one or more drugs at different drugs or different concentrations. The method 101 includes a step of incubating the specimen in the wells 115 to allow differential growth in response to the different drugs or different concentrations of drugs. Finally, individual cells of a particular species are counted 121 in each well to identify the drug or drug concentration that inhibits the growth of the particular species. Important points about the method for antimicrobial susceptibility testing are: (i) no cell / colony purification; (ii) polymicrobial sample; (iii) differential growth; (iv) species-specific detection; and (v) counting of individual cells. No purification of cells or colonies is included, which also means that the specimen, be it blood or another bodily fluid, may be directly input into the method, the raw specimen being divided into wells 109 and incubated 115 with different drugs or concentrations of one or more drugs. The method 101 is refractory to polymicrobial samples and provides species-specific results regardless of the presence of other microorganisms. Species-specific detection operates via species-specific probes, such as fluorescently labeled oligos, that hybridize with the rRNA of a particular species. Differential growth means that cells of a particular species grow differently in different wells depending on the presence of different antimicrobial drugs or their concentrations. Finally, after labeling, the disclosed method and device is useful for counting individual cells without magnification. The incubating 115 step may include fluorescently labeling the microorganisms in a species-specific manner, and the counting 121 step may include imaging each well and counting the fluorescent spots in the image. The labeling step may use target-specific fluorophore-labeled nucleic acid or nucleic acid analog probes, and the counting step may utilize fluorescent in situ hybridization, notably without the need for nucleic acid extraction, purification, or amplification.In certain embodiments, the steps of dividing 109, incubating 115 and counting 121 are performed in a cartridge containing the wells.

[0129] FIG. 2 illustrates an exemplary cartridge 201 (e.g., analytical cartridge, test device). The cartridge 201 includes a sample well 203 that is connected to a divided well 205 via a channel 215. Each divided well is connectable to a corresponding reagent well 209 by a channel through a sliding valve 207. The reagent wells 209 may contain one or any number of reagents, which may be present in particles 241, which may be, for example, lyophilized beads. Each reagent well 209 may be fluidly connected to a corresponding imaging well 211. One or more imaging wells 211 may contain a dye cushion 215. The cartridge optionally includes a diluent reservoir 221, and when a button 227 is pressed, a diluent (e.g., a growth medium such as trypticase soy broth or saline) flows into the divided well 205. The cartridge may generally be described according to the dimensions of length l, height h, and width w, e.g., l of about 6 cm, h of about 4 cm, and w of about 2 cm. The analytical cartridge 201 can be operated manually or in combination with an instrument (e.g., an analyzer) in which the specimen is exposed to, for example, a species-specific detectable label and a magnetic tag that can together form a quaternary complex with a target in the specimen for imaging. For example, the species-specific detectable label can be any suitable label that is detected by imaging known in the art. In a preferred embodiment, the detectable label is an oligonucleotide probe labeled with a fluorophore complementary to a ribosomal RNA sequence dedicated to a microbial species. The complex can be separated from the unbound label by using a magnetic field on the analytical cartridge 201 to pull the complex to a surface within the cartridge 201. The microbial complex can then be imaged and the image analyzed (manually or by a computing device) to identify the microorganisms present in the specimen. A report may be generated. The report may, for example, provide an identification of the microbial target and / or the image. The report may include a qualitative result ("infections detected"). The report may state whether the result is above or below a threshold level for a panel of pathogen targets.The report may provide an antimicrobial susceptibility profile. In various embodiments, such as microbial identification embodiments, the analytical cartridge 201 may be preloaded with one or more species-specific labels so that a user who suspects that a specimen contains one or more microorganisms may select a preloaded analytical cartridge for microbial identification. In antimicrobial susceptibility testing embodiments, the specimen 105 may be distributed into portions within the cartridge 201, with each portion containing a different reagent, such as a different antimicrobial, or a different concentration of one or more antimicrobials.

[0130] Detectable labels may include fluorescent molecules, radioisotopes, mass tags for mass spectrometry, or chemiluminescent molecules. Detectable labels may include species-specific moieties that preferentially bind to a particular species of microorganism. Species-specific binding molecules may include, for example, antibodies to target-specific molecules or antigens, or oligonucleotide or polynucleotide probes complementary to species-specific nucleic acid sequences. In a preferred embodiment, the detectable labeling of microorganisms includes fluorescent in situ hybridization (FISH). FISH analysis uses fluorescent probes that include a nucleic acid (or nucleic acid analog) probe portion and a fluorophore probe portion that binds via reassociation with a species-specific nucleic acid sequence so that the species of the microorganism can then be optically detected. For example, a probe targeting a species-specific 16S rRNA can be used to selectively tag and detect the microorganism. See Volkhard, et al., 2000, Fluorescent In Situ Hybridization Allows Rapid Identification of Microorganisms in Blood Cultures, J Clin Microbiol., 38(2):830-838, which is incorporated by reference. In a preferred embodiment, the detectable label is a fluorescently labeled probe oligonucleotide complementary to ribosomal RNA specific to a microbial species.

[0131] Species-specific probes are designed according to methods known in the art. For example, species-specific probes may be designed by using software such as ARB, PRIMROSE and mathFISH, and public databases such as probeBASE, RDP and SILVA. The embodiments of the present invention relating to identifying microorganisms in a specimen may include multiple detectable labels, such as separate species-specific fluorescent probes for independently identifying multiple distinct species of microorganisms in a single specimen. The separate fluorescent probes may include separate nucleic acid probe portions designed such that under reassociation conditions of the assay, the nucleic acid probe portions of the fluorescent probes preferentially reassociate with the species-specific nucleic acid sequences of the distinct species of microorganisms.

[0132] The fluorophores on the separate fluorescent probes can have separate photon signals so that the fluorescent signals for different species of microorganisms in the assay can be distinguished by their separate photon signals. Imaging methods for identifying multiple separate photon signals are known to those skilled in the art. For example, multiple images can be acquired using separate pairs of excitation and emission optical filters that correspond to the action spectra of the separate fluorophores. Thus, portions of a single specimen can be tested for the presence of multiple specific target cells or microorganisms in a single treatment and imaging well. The magnetic tag can be any known in the art, including paramagnetic particles. The target-specific magnetic tag can specifically bind to a class of microorganisms, for example, only bacteria.

[0133] In certain embodiments, identification of microorganisms in a sample is performed by using FISH analysis at a constant temperature, where the analysis does not require fixation of cells, additional reagents are added during the analysis, and no washing is required by the user to remove unbound fluorescent label, thus allowing automated sample processing in a single cartridge with a time from start of the reaction to obtaining imaging results of about 30 minutes or less.

[0134] The magnetic tag and detectable label preferably form a complex with the target cells present in the specimen. A magnetic field can be applied to the magnetic particles to physically separate the bound detectable label from the unbound detectable label in the solution without a washing step. A dye cushion layer, such as that described in U.S. Pat. No. 9,643,180, incorporated herein by reference, can be used in combination with magnetic particles and a magnetic field to attract the microorganisms through a dense, opaque aqueous dye cushion layer and place them on a detection surface in the well of an analytical cartridge for imaging analysis. The dyes in the dye cushion layer are preferably selected to absorb the excitation and emission light used by the instrument for imaging. Thus, the signal from the unbound detectable label on the dye cushion layer ("assay layer") does not significantly interfere with the detection of the signal from the labeled target cells or microbial complexes magnetically placed on the detection surface. Similarly, the use of the dye cushion can prevent autofluorescence from the specimen matrix that may be contained in the assay layer from significantly interfering with the detection of the signal from the placed labeled target cells or microbial complexes. These attributes of the dye cushion may allow for the detection of microorganisms without sample preparation by the user and without a washing step to remove unbound label from the cartridge.

[0135] Non-magnified digital imaging is preferably used to detect the labeled target cells that are placed on the detection surface. In the preferred case of fluorescent labeling, various lenses, illumination sources, excitation sources and filters may be used. The imaging system may include any device that can generate a digital image of the detectably labeled target microorganism in solution or attract it to the detection surface in the well of the cartridge. The imaging system may be contained in a subsystem of the instrument described herein. The imaging system may include, for example, a CCD camera, a CMOS camera, a line scan camera, a CMOS avalanche photodiode (APD), a photodiode array, a photomultiplier tube array, or other types of digital imaging detectors. Imaging may be performed under a single set of conditions or light sources, and filters and / or lenses may be changed between images to detect different optically distinguishable labels (e.g., different fluorescent probes corresponding to different microorganisms). The imaging techniques, analyzers and instruments described in U.S. Pat. Nos. 9,643,180 and 8,021,848, both of which are incorporated by reference, may allow for the identification and enumeration of individual microorganisms or other target cells.

[0136] FIG. 3 is a schematic diagram of the steps of another exemplary method 301 of the present invention. A patient specimen 105 (e.g., a polymicrobial specimen) may be directly introduced 107 into the analytical cartridge 201, without the need for any pretreatment steps. The analytical cartridge 201 is operable, manually or in combination with an instrument, to divide 111 the specimen 105 into multiple portions 113 (e.g., compartments) within the analytical cartridge 201. One or more portions 113 may not have been exposed to an antimicrobial agent and / or incubated. The leftmost passage shows a portion 113 of the specimen 150 that has not been exposed to an antimicrobial agent and / or incubated, and is imaged 123 to establish the number of target cells present in that portion prior to growth incubation ("time 0 count"). The portions 113 may be incubated in the presence of different antimicrobial agents 117, which may already be present in the analytical cartridge 201, and may be selected based on target cells or microorganisms suspected or known to be present in the specimen 105. For example, species of bacteria such as bacteria associated with urinary tract infections (eg, Escherichia spp. (eg, E. coli), Klebsiella spp., Enterococcus spp., or Pseudomonas spp.) may be suspected to be present in the specimen.

[0137] The specimen 105 may be mixed with an amount of growth medium in the cartridge 201 at any time during the introduction 107 and incubation 115 of the specimen 105 into the analytical cartridge 201. The incubation 115 step may continue for a suitable amount of time, such as about 4 hours, to allow for measurable differential growth of the microorganisms in the specimen 105. The growth medium and / or antimicrobial agent may be selected and included in the cartridge 201 based on the microorganisms to be analyzed in the specimen 105. For example, a growth medium known to support the growth of the identified microorganism, and a therapeutic or antimicrobial agent typically used to treat the identified microorganism may be selected. In a preferred embodiment, the analytical cartridge may be preloaded with a growth medium and therapeutic agent for a particular target, such that a user with an identified microorganism in a specimen (e.g., determining the origin of a patient's infection) may select an appropriate prepackaged analytical cartridge for antimicrobial susceptibility testing analysis of the identified target microorganism.

[0138] After incubation 115, the incubated portions 117 may be exposed to, for example, species-specific magnetic tags and detectable labels that can form complexes 119 with specific microorganisms in the portions for imaging 123. The microorganism 121 complexes can then be imaged 123 in the portions and the images 125 analyzed 127 (manually or by a computing device) to quantify the amount of microorganism 121 present in each portion. By comparing the amount of microorganisms in portions incubated in the presence of various antimicrobial agents, it can be determined which of those agents inhibit growth. The comparison preferably includes the amount of target cells from a "time zero count" growth reference portion that was split from the original specimen, processed, and imaged without incubation. A report 129 may be generated that may include the results of the analysis 127 and may include the images 125 and recommendations for treatment.

[0139] Target microorganisms and cells contemplated for testing using the system and method of the present invention include viruses or bacteria. The target microorganisms are preferentially those that usually cause the particular type of infection being investigated. For example, for urinary tract infections, the target pathogens may include Bacillus antracis, Clostridium difficile, Neisseria gonorrhoeae, Mycobacterium tuberculosis, Acinetobacter baumannii, E. coli, Klebsiella pneumoniae or Pseudomonas aeruginosa, to name a few.

[0140] In the case of bacterial targets, for example, the specimen can be incubated in the presence of various antibacterial agents, and the effect of these agents on the growth of the target bacteria in the specimen can be analyzed to determine efficacy. The systems and methods described herein can be similarly applied to measure the cytotoxic effect of chemotherapeutic agents on cancer cells, or the effectiveness of various antiviral agents on the viral load in a specimen. The only variations that may be required across different target cells and microorganisms are the target-specific binding molecules (e.g., bacterial cell surface-specific antibodies, viral-specific oligonucleotide probes, or cell-specific dyes), the treatments being tested (e.g., antiviral drugs, antibacterial agents, or cancer therapies), the incubation time, and in some cases, the characteristics being analyzed in the image analysis (e.g., CFU quantification, viral load, or cell number, morphology, or function).

[0141] The cartridge 201 includes an inlet or sample well 203 for receiving a specimen, a distribution well 205, a reagent well 209, an imaging well 211, and a channel 213 for the specimen to move between the wells, as well as a valve 207 for controlling the movement. The cartridge 201, when compatible with an instrument such as an analyzer, is operable to receive a specimen in the sample well 203 and split the specimen into portions in the distribution well 205 via the channel 213. The valve 207 can control the movement of fluid between the distribution well 205 and the reagent well 209 and the imaging well 211. In a microorganism identification application of the present invention, the valve 207 may be configured to allow one or more portions to bypass the distribution well 205 and to proceed directly to one or more of the reagent wells 209 and the imaging well 211. The reagent well 209 may be pre-filled with a reagent for labeling the microorganism for imaging. The labeling reagent may be lyophilized for storage and activated upon contact with the specimen portion of the fluid. Reagent well 209 may contain, for example, a detectable label and a magnetic tag, such that when a sample portion passes through reagent well 209, a microorganism-specific complex is formed comprising the target microorganism, the detectable label and the magnetic tag.

[0142] In other embodiments of the invention, valve 207 may also be configured to allow one or more portions to bypass distribution well 205 and proceed directly to one or more of reagent wells 209 and imaging well 211 to provide a zero growth reference or baseline for antimicrobial susceptibility testing. Distribution wells 205 can be pre-filled with growth medium and / or one or more antimicrobial agents. Valve 207 can retain specimen portions in distribution wells 205 for incubation for any period of time in the presence of various antimicrobial agents.

[0143] FIG. 4 shows an exemplary instrument 601 (e.g., analyzer) for performing microbial identification and antimicrobial susceptibility testing of specimens in cartridge 201. The instrument 601 may be used to interact with cartridge 201 to perform target cell identification and antimicrobial susceptibility testing of the specimen. The instrument 601 includes at least one user interface 603 (e.g., touch screen) for displaying prompts, results, reports, and for receiving commands. The instrument 601 may include different functional areas and subsystems. These may include a carousel for transporting and incubating the analytical cartridge; a fluidic subsystem that provides a pneumatic interface with the cartridge fluids; a magnetic; an upper portion 607 for housing processing and incubation equipment; and a lower portion 609 for housing electronics, imaging, and pneumatic equipment.

[0144] 5 is a top view of the components within the upper portion 607 of the instrument 601, showing the carousel that aids in moving the cartridges 201 around the instrument 601. The instrument 601 may include a loading tray 703 for receiving and sorting multiple assay cartridges 201.

[0145] The instrument 601 may also include a carousel 605 for accepting, moving, and manipulating the analytical cartridges within the instrument 601, and a mechanical cartridge transporter 707. The instrument 601 may also include a task scheduler. The instrument 601 is preferably controlled by a computer that automates the manipulation of the analytical cartridges, the performance of microbial identification and antimicrobial susceptibility testing, and the generation of results. The instrument 601 may include multiple subsystems for performing the methods of the present invention.

[0146] The subsystems of the instrument 601 may include a pneumatic subsystem 709, a magnetic subsystem 711, an imaging subsystem 713, and a waste subsystem 715 (e.g., a disposable bin into which a used cartridge 201 can be pushed). The magnetic subsystem 711 may include, for example, permanent magnets and electromagnets that provide a magnetic field to place complexes of magnetic particles and targets on the detection surface of the analytical cartridge for imaging. The imaging subsystem 713 may be such as those described in U.S. Pat. Nos. 9,643,180 and 8,021,848, both of which are incorporated herein by reference, for capturing images of microorganisms, as well as a stage for manipulating the detection surface of the analytical cartridge 201 relative to the imaging module of the instrument 601. The imaging subsystem 713 may be operatively associated with a computer that provides image processing, analysis, and display capabilities. The pneumatic subsystem 709 may be operable to drive movement of the specimens 105 and reagents within the analytical cartridge 201, for example, via manipulation of valves 207 (e.g., plungers and actuators) using functionality provided by air pressure or vacuum. In some embodiments, hydraulic or mechanical means may also be incorporated into the pneumatic subsystem 709 to effect movement of the specimen 105. The waste subsystem 715 may include a container (e.g., a removable bin) for disposal of the cartridge 201 after use.

[0147] The instrument 601 may also include one or more incubation areas for holding (or storing) the analytical cartridges during growth incubation and / or assay incubation. The incubation areas may include heating and / or cooling elements and thermostat controlling elements for maintaining the incubation areas at a desired temperature (e.g., 35° C.) for growth of target cells or microorganisms or for performing assay incubation.

[0148] In some embodiments, the mechanical cartridge carrier 707 (e.g., a mechanical conveyor arm) may be operable to manipulate the analytical cartridge 201 among various subsystems within the instrument 601. In some embodiments of the invention, the mechanical cartridge carrier 707 moves each cartridge 201 between the carousel 605 and the various subsystems of the instrument. The mechanical cartridge carrier 707 applies a pushing force to move the cartridge 201 to and off the carousel 605. The carousel 605 may rotate to a position of the analytical cartridge 201 adjacent to another one of the subsystems, and the mechanical cartridge carrier 707 may then apply a force to slide the analytical cartridge 201 on the subsystem. The cartridge 201 is never picked up by the mechanical cartridge carrier 707 or any other element of the instrument 601. Sliding or pushing the analytical cartridge 201 within the instrument 601 reduces exposure to debris.

[0149] In some embodiments, the instrument includes a task scheduler for managing the cartridges 201 within the instrument 601. The task scheduler is operable to control the movement, such as transportation and movement, of each cartridge 201 among multiple subsystems. In some embodiments, the time that each analytical cartridge 201 spends in the subsystems may also be managed by the task scheduler. The task scheduler may reserve time in the various subsystems required for the analysis of each cartridge 201. In some embodiments of the invention, the task scheduler may manage the movement of the analytical cartridge 201 (i.e., the steps / parameters of the analysis to be performed) by identifying the contents of the cartridge.

[0150] In some embodiments, the instrument 601 may also include a reader operable to analyze a unique identifier (e.g., a barcode) 219 located on the analytical cartridge 201. The contents of the analytical cartridge 201 and the required processing may be associated with the unique identifier 219 on the analytical cartridge 201. Each cartridge 201 may include a unique identifier readable by the instrument 601. The instrument 601 may read the unique identifier 219 via the reader and associate the unique identifier 219 with a particular set of instructions for the task scheduler to execute. The reader may be located outside the instrument 601 and may read the unique identifier 219 before the analytical cartridge 201 moves from the loading rack 703 to the carousel 605. The unique identifier 219 may be a barcode and may be specific / unique to the analyte in the analytical cartridge 201.

[0151] The specimen may be inserted directly into the sample well 203. The air port 217 may be opened to apply pressure or vacuum to distribute the specimen into the wells in the analytical cartridge 201. The specimen may first be distributed from the sample well 203 to the distribution well 203, held there by the valve 207 during incubation, and released into the reagent well 209 for labeling and then into the imaging well 211 for imaging. The analytical cartridge 201 has a unique identifier 219 (e.g., a barcode) associated with the cartridge that, when analyzed or read by the instrument or a reader in the instrument, carries with it a set of instructions for processing within the instrument.

[0152] The cartridge 201 can then be subjected to a magnetic field (e.g., placed on a permanent magnet) to attract the complexes to the detection surface 215 at the bottom of the imaging well 211. The imaging well 211 may contain a dye cushion that forms a dense, opaque aqueous layer below the upper layer (containing the biological material, detectable label, complexes, magnetic tags) in the imaging well 211 as described herein. The complexed magnetic particles are pushed through the lower dye cushion layer and deposited on the detection surface 215 of the imaging well 211 by the magnetic field that attracts the complexes and the microbial complexes through the dye cushion layer to the detection surface 215 on the imaging well 211. The detection surface 215 may be optically clear to allow optical detection of the microorganisms. After processing and pulling down with the magnetic field, the cartridge 201 can be placed in an imaging system for image processing as described herein.

[0153] Species-specific fluorescent oligonucleotide probes may be used to fluorescently label target microorganisms such that imaging of the fluorescent label provides identification and quantification of the target microorganism (e.g., cell counting of target bacteria in a specimen). The analytical cartridge can have any number of distribution wells, as well as corresponding reagent and imaging wells. In a preferred embodiment, the analytical cartridge has a "time 0 count" reference portion, and the differential growth analysis of one or more antimicrobial agents is present at at least two separate concentrations, respectively. In some embodiments, the concentrations selected should preferably correspond to the breakpoint concentrations of accepted antimicrobial susceptibility testing established by CLSI and / or EUCAST for a particular target species and antimicrobial agent.

[0154] Although the system and method of the present invention are described herein primarily in the context of an analytical cartridge with interconnected wells for automated processing, the technology can also be performed in any known fluid platform, including manual processing in the wells of a microtiter plate or on a substrate where droplets function similarly. In some embodiments, the cartridge contains at least 8 wells and can be made of two parts containing at least 16 wells, which are made up of two 8-well sections. However, the number of wells is preferably large (e.g., 100 or more), limited only by the size limitations of the cartridge and the instrument.

[0155] By quantifying the amount of target cells or microorganisms present in each specimen portion after incubation in the presence of various antimicrobial agents, and by comparison with a "time 0 count" reference determined before incubation, the effect of each antimicrobial agent on target cell replication can be determined. By determining which antimicrobial agent most inhibits growth, an effective therapy for treating the patient's infection can be determined.

[0156] FIG. 6 is an example of a workflow for performing antimicrobial susceptibility testing of a specimen containing bacteria. The workflow is an exemplary incubation and processing step that may be performed in an analytical cartridge according to various inventive methods. The specimen may be mixed with a growth medium 501, which may be selected based on knowledge of the target microorganism present in the specimen (e.g., cation-adjusted Mueller Hinton Broth selected for analysis of E. coli in the specimen). The specimen and growth medium may then be divided 503 into many (n) distribution wells for incubation 507. One or more distribution wells may contain various antimicrobial agents. One or more portions may be analyzed 505 without incubation (t0 specimen) to quantify the target microorganism for subsequent comparison to specimen portions analyzed after incubation in the presence or absence of an antimicrobial agent. Incubation in the absence of a therapeutic agent may provide important information, including an indication of non-viable microorganisms, which may occur, for example, if the patient has already been treated with an antimicrobial agent. Analysis of such incubated portions may also serve as an internal control for prevention of growth inhibition and stability of the reagent.

[0157] Preferably, bacteria or other target cells have already been identified, and various antimicrobial agents, such as antimicrobial agents commonly used to treat the identified bacteria, have been selected accordingly. Each distribution well may contain a single antimicrobial agent or a unique combination of antimicrobial agents to evaluate the combined effect on target cell growth. Different concentrations of different antimicrobial agents may be combined with specimen portions or aliquots in different distribution wells, and some distribution wells may be used to replicate treatment with the same antimicrobial agent to enhance results. Specimen portions or aliquots in some distribution wells may not be combined with antimicrobial agents to quantify uninhibited growth and / or detect growth inhibition due to either non-antimicrobial inference or reagent instability.

[0158] After incubation 507, the incubated specimen aliquots are processed and imaged in a similar manner as the "Time 0 Count" specimen aliquots 505, and bacterial and other microbial counts can be compared to one another, and the "Time 0 Count" specimen aliquots, or other controls, are counted 509 to determine the effectiveness of a particular concentration or combination of antimicrobial agents. Preferably, antibiotics are recommended for treatment at concentrations that do not permit growth.

[0159] 7 illustrates the step of a user directly transferring a specimen 105 into a sample well 203 of an analytical cartridge 201. In this example, the specimen 105 is pipetted from a specimen collection device 1001 (e.g., a specimen collection container, swab, etc.) into the sample well 203 of the analytical cartridge 201. In other embodiments, the specimen collection device 1001 (e.g., a swab) may be inserted directly into the sample well 203 of the analytical cartridge 201.

[0160] FIG. 8 depicts a number of cartridges 201 being loaded into a loading tray 703 prior to being transported to the instrument 601 .

[0161] The instrument may include or be connected to a computer useful for performing the steps of the method. The computer may be operable to control the instrument 601 and the cartridge 201 and / or to process the results of the imaging. The computer may include a processor coupled to a non-transitory memory device. The memory preferably stores instructions executable by the processor to cause the system to manipulate the cartridge 201 in the instrument 601 and to obtain and process images of the labeled microorganisms.

[0162] A processor refers to any device or system of devices that performs processing operations. A processor generally includes a chip, e.g., a single-core or multi-core chip, to provide a central processing unit (CPU). The process may be provided by a chip from Intel or AMD. The processor may be any suitable processor, such as the microprocessor sold under the trademark XEON E7 by Intel (Santa Clara, CA) or the microprocessor sold under the trademark OPTERON 6200 by AMD (Sunnyvale, CA).

[0163] Memory refers to a device or system of devices that stores data or instructions in a machine-readable format. The memory may include one or more sets of instructions (e.g., software) that, when executed by one or more processors of the computer, can accomplish some or all of the methods or functions described herein. Preferably, the computer includes non-transitory memory such as a solid-state drive, flash drive, disk drive, hard drive, subscriber identity module (SIM) card, secure digital card (SD card), micro SD card, or solid-state drive (SSD), optical and magnetic media, etc., or combinations thereof. In a preferred embodiment, the memory includes task scheduler software. Different task scheduler instructions may be associated with specific unique identifiers. Scanning of a unique identifier on the analysis cartridge 201 or in the instrument 601 via a reader or barcode scanner may cause the processor to execute task scheduler instructions specific to the unique identifier.

[0164] The input / output device 603 is a mechanism or system for transferring data to or from a computer. Exemplary input / output devices, such as a touch screen, include a video display unit (e.g., a liquid crystal display (LCD)), an alphanumeric input device (e.g., a keyboard), a cursor control device (e.g., a mouse), a barcode scanner, a reader, a disk drive unit, a signal generating device (e.g., a speaker), an accelerometer, a microphone, a cellular radio frequency antenna, and a network interface device, which may be, for example, a network interface card (NIC), a Wi-Fi card, or a cellular modem. The input / output device may be used to allow a user to control the instrument 601, display results, and generate reports resulting from the analysis of cartridges received by the instrument 601. EXAMPLES

[0165] Example A Identification of urinary tract infections (UTIs) and antimicrobial susceptibility testing / minimum inhibitory concentration assays using microtiter plates

[0166] Analysis of urine specimens was performed to determine the presence of a urinary tract infection, whether the patient was infected with one of the four most common UTI causative organisms (i.e., E. coli, K. pneumoniae, Enterococcus spp., and P. aeruginosa), and which of the four major antimicrobial agents were effective in treating the UTI. Urine was added directly to the analytical cartridge of the present invention for testing and to growth medium for incubation within the cartridge. If positive for one of the four UTI organisms, antimicrobial susceptibility testing was then performed. Identification results were obtained within 30 minutes, and antimicrobial susceptibility testing results were obtained within 4 hours. Minimum inhibitory concentration (MIC) analysis was performed using the exemplary technology of the present invention.

[0167] Urine specimens were tested for the presence of E. coli, K. pneumoniae, Enterococcus spp. and P. aeruginosa, each with a detection limit less than or equal to 10,000 CFU / mL (a common threshold for diagnosing UTI). Species-specific labels were used. In this example, all four target microorganisms were detected within 30 minutes using an isothermal FISH reaction and a common reaction diluent. Detection of all strains (10 E. coli, 11 Klebsiella spp., 10 P. aeruginosa and 6 Enterococcus spp.) in each specimen was achieved without off-target detection. Cross-reactivity of the FISH probes was carefully analyzed for E. coli, K. pneumoniae, Enterococcus spp. and P. aeruginosa against 15, 10, 10 and 10 different species of bacteria, and no cross-reactivity was observed.

[0168] The limit of detection (LoD) results using 30% urine spiked with the microorganisms E. coli, K. pneumoniae, Enterococcus spp. and P. aeruginosa across the four specimen types are shown in Table 1 and indicate high analytical sensitivity for UTI causative microorganisms.

[0169] FIG. 9 shows the number of E. coli cells counted using the disclosed instruments, methods and cartridges to analyze urine spiked samples with different numbers of colony forming units (CFU) per mL.

[0170] FIG. 10 shows the number of Enterococcus faecalis cells enumerated using the disclosed instruments, methods and cartridges to analyze urine spiked samples with different numbers of colony forming units (CFU) per mL.

[0171] The microbial detection and identification methods described herein can be used to rapidly detect other species of microorganisms, such as Bacillus antracis (B. antracis) and Clostridium difficile (C. difficile or C. diff).

[0172] [Table 1]

[0173] In a separate experiment, 7-10 strains of each of the four identified microorganisms were subjected to AST analysis for common antimicrobials using both the exemplary method of the present invention and the conventional broth microdilution method, and the results were then compared.

[0174] Figure 11, for example, gives specific results for the ATCC 2469 strain of E. coli for nitrofurantoin. The results of the essential agreement between the conventional method and the method of the present invention are shown in Table 2 below.

[0175] [Table 2-1] [Table 2-2]

[0176] To test robustness to variable inoculum levels, inocula covering four orders of magnitude were tested using rapid AST methods and compared with known MIC results. As shown in Table 3, 100% essential agreement was observed for all analytes tested (three for four different antimicrobials).

[0177] [Table 3]

[0178] Figure 12 presents results across a range of inoculum concentrations for nitrofurantoin MIC testing. The impact of polymicrobial specimens was tested by spiking the target E. coli with non-target bacteria including Staphylococcus epidermidis, Micrococcus luteus, Corynebacterium minutissimum, Staphylococcus aureus, Acinetobacter baumannii, Citrobacter freundii, and Klebsiella pneumoniae secreting NDM1. Essential agreement of 100%, 100% and 96%, respectively, was observed in MIC testing of five different antimicrobial agents using high concentrations of 1e5, 1e6 and 1e7 CFU / ml of non-target bacteria. Essential agreement of greater than 98% was observed across 84 tested specimens. The E. coli MIC for imipenem was not affected in the presence of 1E7 CFU / mL of K. pneumoniae NDM1-secreted carbapenemase.

[0179] Example B Antimicrobial susceptibility testing: E. coli

[0180] Urine specimens were spiked with clofazimine-susceptible and clofazimine-resistant strains of E. coli. The specimens were tested using an exemplary system (analytical cartridge and instrument) described herein. The specimens were incubated in 32 μg / mL clofazimine for 4 hours.

[0181] FIG. 13 depicts growth results demonstrating that the methods and systems of the present disclosure successfully differentiate between clofazimine-susceptible and clofazimine-resistant strains of E. coli as described herein.

[0182] Example 1 Limits of detection (LoD) for Gram-negative bacteria using a novel rapid fluorescent in situ hybridization assay

[0183] Overview: The following examples demonstrate that very low concentrations of cells can be detected using a novel isothermal fluorescence in situ hybridization method. Detection limits are shown for three common human urinary tract infection (UTI) pathogens. Testing Method:

[0184] Bacterial cell preparation: Bacterial cultures for E. coli ATCC 19138, K. pneumoniae ATCC 700603 and P. aeruginosa ATCC 9721 were obtained by inoculating Trypticase Soy Broth (TSB, Hardy Diagnostics Cat. U65) with 3-5 colonies from a fresh Tryptic Soy Agar plate (TSA, BD Cat. 221185) and growing at 35°C for 1.5-3 hours to achieve log phase growth. After the cells reached an optical density reading at 600 nm of 0.15-0.30, the cells were placed on ice for at least 15 minutes and then diluted. After cooling, cells were diluted in 1x cation-adjusted Mueller-Hinton broth (MHBII, Teknova catalog M5860) to the concentrations to be assayed (approximately 19200, 9600, 4800, 2400, 1200, 600, 300 and 150 colony forming units (CFU) / reaction). For more accurate cell concentrations, these estimated bacterial inputs were adjusted using colony counts. Plate counts were determined by diluting log phase cultures in MHBII to approximately 500 CFU / mL, plating 100 μL onto TSA plates and counting colonies after 16-24 hours of growth at 35°C. The average plate counts were used to calculate the actual CFU present in each concentration tested.

[0185] Preparation of magnetic particles: Polyaspartic acid conjugated magnetic particles (Fluidmag-PAA, Chemicell, catalog 4108) and carboxyl-coated high iron magnetic particles (Carboxyl Magnetic Particles, Spherotech, catalog CM-025-10H) were used to nonspecifically capture bacterial cells. Each particle was added at approximately 1.38 × 10 per reaction for polyaspartic acid particles. 9 particles, 3.46 × 10 per reaction for carboxyl particles. 9 The final concentration of particles was diluted 1:40 in 50 mM Epps buffer, pH 8.2. Fluorescent magnetic microspheres containing green dye (Dragon Green Fluorescent Microspheres, BANGS Laboratories, Cat. MEDG001) were added at 3 × 10 6 The magnetic particles were added to the suspension at a final concentration of 10 ...

[0186] Preparation of FISH probes: Two species-specific DNA oligomer sets for E. coli and K. pneumoniae and one species-specific DNA oligomer set for P. aeruginosa were heated in a water bath between 80-85°C for 10 min and then placed on ice to reduce aggregation. The DNA oligomer sets included species-specific DNA oligonucleotides labeled with a fluorescent dye (Alexa647N, Thermo Fischer) either at the 5' end or at both the 5' and 3' ends of the oligonucleotide, as well as two to six helper oligonucleotides that bind adjacent to or near the specific probes and are designed to disrupt the local secondary structure of the ribosomal subunits and allow for higher hybridization efficiency of the labeled specific probes to the target rRNA. The probe sequences used in this example are shown in Table A in Figure 17.

[0187] Preparation of dried hybridization buffer plate: A mixture of 10x SSC (1.5M NaCl, 0.15M sodium citrate, Sigma, catalog S6639), 2.6% w / v CHAPSO (Sigma catalog C3649), 2.4% w / v SB3-12 (Sigma catalog D0431), 0.43M guanidine thiocyanate (Sigma catalog G9277) and 0.6% w / v cetrimide (Sigma catalog M7365) was prepared. 30 uL of this mixture was added to each well of a 96-well plate. The plate was placed in a 50°C convection oven and allowed to dry overnight. Addition of 100 uL of liquid to these wells achieves the correct hybridization buffer concentrations of 3x SSC (0.45M NaCl, 0.045M Sodium Citrate), 0.77% w / v CHAPSO (Sigma Catalogue C3649), 0.72% w / v SB3-12 (Sigma Catalogue D0431), 0.13M Guanidine Thiocyanate (Sigma Catalogue G9277) and 0.18% w / v Cetrimide (Sigma Catalogue M7365).

[0188] Limit of Detection (LoD) Assay Procedure: A mixture of DNA oligonucleotide sets appropriate for the bacteria of interest was combined with urine and Mueller Hinton Stock adjusted with concentrated cations (MHBII) to make a final solution containing 1x MHBII and 30% pooled human urine (Innovative Research, Cat. IRHUURE500ML). Probe concentrations varied between different bacterial species but ranged from 0.2-0.6 μM for the labeled oligonucleotides and 1.5-6 μM for the corresponding helper probes. 90 uL of this mixture was placed in the appropriate dried hybridization buffer plate. 10 uL of magnetic particle mixture was added followed by 10 uL of the appropriate cell dilution. Twelve replicates of each cell concentration and 24 replicates of blanks (medium without bacteria) were evaluated for each target bacterium tested. 100 μL of the final reaction mixture was transferred to a microtiter plate containing 50 μL (pre-dried) per well of "dye cushion" (50 mM TRIS pH 7.5 (Sigma Cat. T1075), 7.5% v / v Optiprep (Sigma Cat. D1556), 50 mg / mL Direct Black 19 (Orient Cat. 191L)) and incubated at 35° C. for 30 min to allow for simultaneous rehydration of the "dye cushion", labeling of the bacterial cells, and binding of the magnetic particles to the bacterial cell surface. After incubation, the microtiter plate was placed on a magnetic field (Dexter magnetic technologies, Cat. 54170260) for 4 min to bring the magnetic particles, the fraction containing the labeled cells, in close proximity to the imaging surface at the bottom of the well via the "dye cushion".

[0189] Imaging of Labeled Cells: The MultiPath™ Laboratory Imaging System is a custom instrument and software capable of automatically capturing image data from selected wells of a microtiter plate. It uses a high-precision linear stage from Prior Scientific (Rockland, MA) to position each well on a fluorescence-based image acquisition subsystem. The instrument is capable of imaging in four separate color channels and uses an objective lens, illumination LEDs, a fluorescence filter set, and a camera. The objective lens has a field of view designed to capture an image of the entire microtiter plate well. The illumination module light source consists of two high-power LEDs per color channel. A series of fluorescent image frames are captured by the camera using a 3.1MP Sony IMX265 monochrome sensor with 12 bits per pixel quantization. A final image for each well is then formed by summing multiple frames. 16 frames were captured with a 100 ms exposure using a 635 / 25 nm excitation and 680 / 40 nm emission filter. The focus particle is imaged with 470 / 40 nm excitation and 520 / 40 nm excitation filters, capturing 2 frames with a 20 ms exposure.

[0190] Data analysis: The number of fluorescent objects detected at each cell concentration was determined. Data from all eight cell concentrations were fitted to a linear regression line and the slope, intercept and standard deviation of the lowest three cell inputs were used to determine the upper limit of blank (LoB) and limit of detection (LoD) for each bacterium tested. result:

[0191] All three bacteria tested showed low limits of detection. Figures 14, 15 and 16 show data generated for E. coli, K. pneumoniae and P. aeruginosa using the linear fits used to calculate the LoB and LoD. The LoB and LoD are shown in terms of detectable CFU in a single reaction well.

[0192] Conclusion: The novel and rapid FISH method described in this example is shown to be a highly sensitive method, with a limit of detection of approximately 500 CFU or less per reaction, using minimally treated urine matrix.

[0193] Variations. This example is illustrative of the performance of this novel FISH method and is not limited to the specific details included in the description. Thus, those skilled in the art will easily understand that many variations are possible, including using different probe sequences and nucleic acid structures (PNA, LNA, etc.), alternative assay chemistries (different detergents, chaotropes, fluorophores, buffers, pH, temperature, reaction time, component concentrations), urine concentrations and urine processing procedures. This methodology can obviously be extended to other biological specimens and other bacterial and non-bacterial pathogens.

[0194] Example 2 Comprehensive: Detecting and identifying different strains of bacterial species using the rapid FISH method of the present invention

[0195] Overview. This example demonstrates the use of the present invention to detect different strains of targeted bacterial species. Raw data for 11 different E. coli strains is shown, and data for K. pneumoniae, P. aeruginosa, P. mirabilis and Enterococcus spp. are summarized. Bacterial cell targets were labeled in 30 minutes using isothermal fluorescence in situ hybridization (FISH) and detected with a MultiPath™ CCD camera-based detection system. Experimental method.

[0196] Bacterial cell preparation: Bacterial cultures for the different strains were obtained by inoculating Trypticase Soy Broth (TSB, Hardy Diagnostics catalog U65) with 3-5 colonies from a fresh Tryptic Soy Agar plate (TSA, BD catalog 221185) and growing at 35°C for 1.5-3 hours to achieve log phase growth. Using optical density at 600 nm to estimate cell concentration, cells were diluted in 1x cation adjusted Mueller-Hinton Broth (MHBII, Teknova catalog M5860) to approximately 600 and 3000 CFU per reaction. For more accurate percent cell detection calculations, these estimated bacterial inputs were adjusted using colony counts. Plate counts were determined by diluting log phase cultures in MHBII to approximately 500 CFU / mL, plating 100 μL onto TSA plates, and counting colonies after 16-24 hours of growth at 35°C.

[0197] Preparation of magnetic particles: Polyaspartic acid conjugated magnetic particles (Fluidmag-PAA, Chemicell, catalog 4108), used for non-specific capture of bacterial cells, were diluted to 2.75 × 10 in 50 mM EPPS buffer, pH 8.2. 12 Fluorescent magnetic microspheres containing green dye (Dragon Green Fluorescent Microspheres, BANGS Laboratories, catalog MEDG001) were diluted 1:20 to 3 × 10 6 The magnetic particles were added to the suspension at a final concentration of 10 ...

[0198] Labeling of bacterial cells: A 100 μL labeling reaction was prepared by mixing diluted cells, isothermal hybridization buffer (0.9× MHBII, 3× SSC (1.5 M NaCl, 0.15 M sodium citrate, Sigma, catalog S6639), 0.77% w / v CHAPSO (Sigma catalog C3649), 0.72% w / v SB3-12 (Sigma catalog D0431), 0.13 M guanidine thiocyanate (Sigma catalog G9277), 0.18% w / v cetrimide (Sigma catalog M7365)), species-specific Alexa647N-labeled DNA or LNA-containing DNA probes targeted to 16S or 23S bacterial rRNA (Integrated DNA Technologies, IDT), helper probes to promote efficient hybridization (IDT) and 30 μL of pooled human urine (Innovative The probe sequences are shown in the table in FIG.

[0199] Urine was first processed through a Zeba 7K MWCO spin column (Thermo Fisher, catalog 89893 or 89892 depending on urine volume) according to the manufacturer's instructions. 10 μL of magnetic particle preparation was then added to this mixture. The final reaction mixture was transferred to a microtiter plate containing 50 μL (pre-dried) "dye cushion" (50 mM TRIS pH 7.5 (Teknova catalog T1075), 7.5% v / v Optiprep (Sigma catalog D1556), 50 mg / mL Direct Black 19 (Orient catalog 191L)) and incubated at 35° C. for 30 minutes to allow simultaneous rehydration of the "dye cushion", labeling of bacterial cells, and binding of magnetic particles to the bacterial cell surface. After incubation, the microtiter plate was placed on a magnetic field (Dexter magnetic technologies, catalog 54170260) for 4 minutes to bring the magnetic particles, the fraction containing the labeled cells, into close proximity with the imaging surface at the bottom of the wells via a "dye cushion."

[0200] Imaging of Labeled Cells: Labeled bacterial cells on the MultiPath Laboratory Imaging System is a custom-built instrument and software capable of automatically capturing image data from selected wells of a microtiter plate. It uses a high-precision linear stage from Prior Scientific (Rockland, MA) to position each well on a fluorescence-based image acquisition subsystem. The instrument is capable of imaging in four separate color channels and uses an objective lens, illumination LEDs, a fluorescence filter set, and a camera. The objective lens has a field of view designed to capture an image of the entire microtiter plate well. The illumination module light source consists of two high-power LEDs per color channel. A series of fluorescent image frames are captured by the camera using a 3.1MP Sony IMX265 monochrome sensor with 12 bits per pixel quantization. A final image for each well is then formed by summing multiple frames. 16 frames were captured with a 100 ms exposure using a 635 / 25 nm excitation and 680 / 40 nm emission filter. The focal particle is imaged with 470 / 40 nm excitation and 520 / 40 nm excitation filters, capturing 2 frames with a 20 ms exposure.

[0201] Data analysis: For each bacterium, the number of fluorescent objects was determined (assay signal). A bacterial strain was considered detected if a signal was detected that was greater than 3 standard deviations above the signal in the cell-free condition.

[0202] Results. Figure 18 shows the assay signal for eleven E. coli strains. All eleven strains were detected above background "no cells" conditions for a cell input of approximately 600 CFU per assay. Figure 19 shows the percentage of cells detected (total assay signal in cell input wells - background assay signal / total cell input). *Data presented are presented as percentile (%) = 100. Although detection efficiency was somewhat variable from strain to strain, this did not inhibit the ability of the assay to detect each of the 11 different E. coli strains.

[0203] The table in Figure 20 summarizes the comprehensiveness results for E. coli, K. pneumoniae, P. aeruginosa, P. mirabilis and Enterococcus spp., which were analyzed in the same manner as E. coli. The strains tested for K. pneumoniae were ATCC 13833, CDC80, CDC44, CDC87, CDC47, CDC43, BAA2470, CDC34, CDC39, ATCC 700603 and BAA-2472. The strains tested for P. aeruginosa were CDC263, CDC242, 9721, CDC236, 27853, BAA-2110, CDC233, 15692, CDC234, CDC246 and CDC261. Strains tested for P. mirabilis were CDC155, CDC29, CDC159, CDC59, ATCC 7002, and CDC156. Strains tested for Enterococcus included ATCC 19433, ATCC 29212, ATCC 33186, ATCC 51575, ATCC 51299, and BAA-2128.

[0204] Conclusions: The novel FISH method described in this example detected all strains tested of five different bacterial species that are among the major pathogens causing clinical symptoms in patients with UTI.

[0205] Variations. This example is illustrative of the performance of this novel FISH method and is not limited to the specific details included in the description. Thus, those skilled in the art will easily understand that many variations are possible, including using different probe sequences and nucleic acid structures (PNA, LNA, etc.), alternative assay chemistries (different detergents, chaotropes, fluorophores, buffers, pH, temperature, reaction time, component concentrations), urine concentrations and urine processing procedures. This methodology can obviously be extended to other biological specimens and other bacterial and non-bacterial pathogens.

[0206] Example 3 Specific detection of target bacteria using rapid isothermal FISH

[0207] Overview. This example demonstrates that the novel isothermal FISH method specifically detects target bacteria without detecting related non-target bacteria, even at very high concentrations. This example shows assay conditions that specifically detect E. coli, but not 16 other bacteria that also cause urinary tract infections (UTIs), have similar rRNA sequences, or are commensals. Experimental method.

[0208] Bacterial cell preparation: Bacterial cultures for the 16 off-target bacteria (listed in Table 1) and E. coli strain ATCC 25922 were grown from single colonies selected from fresh Tryptic Soy Agar plates (TSA, BD catalog 221185), inoculated into Trypticase Soy Broth (TSB, Hardy Diagnostics catalog U65) and grown overnight at 35°C with shaking. 50-80 μL of overnight culture was added into fresh TSB and grown for 1.5-2 hours until the optical density at 600 nm reached 0.15-0.3. Each bacterium was then inoculated at approximately 1 × 10 60 μl into cation-adjusted Mueller Hinton (MHBII, Teknova catalog M5860) at 100 μl. 8 Diluted to cells / mL.

[0209] Selection of bacterial targets to evaluate: Bacterial pathogens to test for specificity were selected for rRNA sequence similarity with the rRNA sequences of the target bacteria, or because they are pathogens commonly found in urinary tract infections (disease targets), and therefore cross-reactivity to these organisms would be most problematic. The table in Figure 22 shows the bacterial species and strains tested.

[0210] Preparation of FISH probes: A DNA probe set for E. coli was heated in a water bath between 80-85°C for 10 minutes and then placed on ice to reduce aggregation. The DNA probe set is shown in the table in Figure 23. The set included species-specific DNA oligonucleotides labeled with a fluorescent dye (Alexa647N, Thermo Fischer) and helper oligonucleotides that bind adjacent to or near the specific probes and are designed to disrupt the local secondary structure of the ribosomal subunits, allowing for higher hybridization efficiency of the labeled specific probes to the target rRNA.

[0211] Preparation of magnetic particles: Polyaspartic acid conjugated magnetic particles (Fluidmag-PAA, Chemicell, catalog 4108), used for non-specific capture of bacterial cells, were diluted to 2.75 × 10 in 50 mM EPPS buffer, pH 8.2. 12 Fluorescent magnetic microspheres containing green dye (Dragon Green Fluorescent Microspheres, BANGS Laboratories, catalog MEDG001) were diluted 1:20 to 3 × 10 6 The magnetic particles were added to the suspension at a final concentration of 10 ...

[0212] Labeling of bacterial cells: A 100 μL labeling reaction was prepared by mixing diluted cells, isothermal hybridization buffer (0.9× MHBII (Teknova catalog M5860), 3× SSC (0.45 M NaCl, 0.045 M sodium citrate, Sigma catalog S6639), 0.77% w / v CHAPSO (Sigma catalog C3649), 0.72% w / v SB3-12 (Sigma catalog D0431), 0.13 M guanidine thiocyanate (Sigma catalog G9277), 0.18% w / v cetrimide (Sigma catalog M7365)), species-specific Alexa647N-labeled probes targeted to 16S or 23S bacterial rRNA (Integrated DNA Technologies, IDT), helper probes to promote efficient hybridization (IDT) and 30 μL of pooled human urine (Innovative A 10 μL magnetic particle preparation was prepared by combining 10 μL of magnetic particle preparation (Thermo Fisher Research, Cat. IRHUURE500ML). The specific probe sets tested are shown in the table in FIG. 23. Urine was first processed through a Zeba 7K MWCO spin column (Thermo Fisher, Cat. 89893 or 89892 depending on urine volume) according to the manufacturer's instructions. 10 μL of magnetic particle preparation was then added to this mixture. The final reaction mixture was transferred to a microtiter plate containing 50 μL (pre-dried) per well of "dye cushion" (50 mM TRIS pH 7.5 (Sigma Cat. T1075), 7.5% v / v Optiprep (Sigma Cat. D1556), 50 mg / mL Direct Black 19 (Orient Cat. 191L)) and incubated at 35° C. for 30 minutes to allow simultaneous rehydration of the "dye cushion", labeling of the bacterial cells, and binding of the magnetic particles to the bacterial cell surface. Each bacterium was added at 1×10 per reaction. 6A final concentration of cells was tested, which is approximately 3000 times higher than the determined detection limit for E. coli. After incubation, the microtiter plate was placed on a magnetic field (Dexter magnetic technologies, catalog 54170260) for 4 minutes to bring the magnetic particles, the fraction containing the labeled cells, into close proximity with the imaging surface at the bottom of the wells via a "dye cushion."

[0213] Imaging of Labeled Cells: The MultiPath™ Laboratory Imaging System is a custom instrument and software capable of automatically capturing image data from selected wells of a microtiter plate. It uses a high-precision linear stage from Prior Scientific (Rockland, MA) to position each well on a fluorescence-based image acquisition subsystem. The instrument is capable of imaging in four separate color channels and uses an objective lens, illumination LEDs, a fluorescence filter set, and a camera. The objective lens has a field of view designed to capture an image of the entire microtiter plate well. The illumination module light source consists of two high-power LEDs per color channel. A series of fluorescent image frames are captured by the camera using a 3.1MP Sony IMX265 monochrome sensor with 12 bits per pixel quantization. A final image for each well is then formed by summing multiple frames. 16 frames were captured with a 100 ms exposure using a 635 / 25 nm excitation and 680 / 40 nm emission filter. The focal particle is imaged with 470 / 40 nm excitation and 520 / 40 nm excitation filters, capturing 2 frames with a 20 ms exposure.

[0214] Data analysis: For each bacterium, the number of fluorescent objects was determined (assay signal). Bacteria were considered cross-reactive if a signal was detected within 3 standard deviations of the signal in the blank (no cells added). result.

[0215] Figures 24 and 25 show that the rapid novel FISH method detects only E. coli and not 16 other different challenge bacteria. Figures 24 and 25 each show that the rapid novel FISH method detects only E. coli and not 16 other different challenge bacteria at very high concentrations (1 x 10 per reaction). 6 The results show that eight clinically relevant challenge bacteria (16 cells) are not detected under the same assay conditions that yield a high assay signal for the targeted E. coli bacteria. The two bars represent two different probe sets designed to be specific for E. coli (see table in Figure 23). The assay signal for each of the 16 challenge bacteria was less than the no cells control plus three standard deviations (125).

[0216] Conclusion: The novel rapid FISH method described in this example, by design, specifically detects E. coli but not 16 clinically relevant potentially cross-reactive bacteria, demonstrating that this method has high specificity for the identification of target UTI pathogens that are of great importance for the clinical treatment of infection.

[0217] Variations. This example is illustrative of the performance of this novel FISH method and is not limited to the specific details included in the description. Thus, those skilled in the art will easily understand that many variations are possible, including using different probe sequences and nucleic acid structures (PNA, LNA, etc.), alternative assay chemistries (different detergents, chaotropes, fluorophores, buffers, pH, temperature, reaction time, component concentrations), urine concentrations and urine processing procedures. This methodology can obviously be extended to other biological specimens and other bacterial and non-bacterial pathogens. Assays have also been designed that demonstrate high specificity for K. pneumoniae, K. oxytoca, P. aeruginosa, P. mirabilis and E. faecalis.

[0218] Example 4 A multiplex FISH method for simultaneously identifying four distinct microorganisms

[0219] Overview: This example demonstrates the use of the present invention to simultaneously detect E. coli, K. pneumoniae, P. aeruginosa, and K. oxytoca in a single reaction using fluorescently labeled probes specific for the rRNA of each bacterium. Each pathogen was specifically detected in the mixture through the use of four separate fluorophores - one for each bacterial species - with distinct excitation / emission spectral characteristics.

[0220] Experimental Methods. Bacterial Cell Growth: Bacterial cultures for Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC 13883, Pseudomonas aeruginosa ATCC 27853, and Klebsiella oxytoca ATCC 8724 were obtained by inoculating Trypticase Soy Broth (TSB, Hardy Diagnostics catalog U65) with 3-5 colonies from a fresh Tryptic Soy Agar plate (TSA, BD catalog 221185) and growing at 37°C for 1.5-3 hours to achieve log phase growth. Each culture was then inoculated into cation-adjusted Mueller-Hinton Broth (MHBII, Teknova, catalog M5860) at approximately 1.0 x 10 8 Dilutions were made to give an optical density at 600 nm of 0.15 colony forming units (CFU) / mL.

[0221] Preparation of magnetic particles: Polyaspartic acid conjugated magnetic particles (Fluidmag-PAA, Chemicell, catalog 4108) and carboxyl coated magnetic particles (Carboxyl Magnetic Particles, Spherotech, catalog CM-025-10H) were used to nonspecifically capture bacterial cells. Each particle was added at approximately 1.38 × 10 per reaction for polyaspartic acid particles. 9 particles, and 3.46 × 10 for carboxyl particles. 9 The mixture was diluted 1:40 into 50 mM Epps buffer, pH 8.2, for a final concentration of 1:

[0222] Labeling of bacterial cells: 100 μL labeling reactions were prepared by combining diluted cells of all four bacteria, isothermal hybridization buffer (0.9× MHBII, 3× SSC (1.5 M NaCl, 0.15 M sodium citrate, Sigma, catalog S6639), 0.77% w / v CHAPSO (Sigma catalog C3649), 0.72% w / v SB3-12 (Sigma catalog D0431), 0.13 M guanidine thiocyanate (Sigma catalog G9277), 0.18% w / v cetrimide (Sigma catalog M7365)), species-specific DNA probes targeted to 16S or 23S bacterial rRNA (Integrated DNA Technologies, IDT), helper probes (IDT) to promote efficient hybridization, and 30 μL of pooled human urine (Innovative Research, catalog IRHUURE500ML). 10 μL of the magnetic particle preparation was then added to the mixture. The probe sequences and the locations of their dye modifications are shown in the table in FIG.

[0223] The cells / hybridization mixture (1 mL) was transferred into the cartridge. The cartridge was placed on the analyzer (described below) which automated the remaining assay steps as well as image acquisition and analysis. Briefly, the analyzer's fluidics system moved a reaction mixture containing 46 μL (pre-dried) per well of "dye cushion" (50 mM TRIS pH 7.5 (Sigma Cat. T1075), 7.5% v / v Optiprep (Sigma Cat. D1556), 50 mg / mL Direct Black 19 (Orient Cat. 191L)) into the optical window. The cartridge was incubated for 30 minutes at 35° C. in the analyzer. After this incubation, the cartridge was moved onto a magnet station (Dexter magnetic technologies, Cat. 54170260) for 4 minutes to bring the magnetic particles, the fraction containing the labeled cells, into close proximity with the imaging surface at the bottom of the well via the rehydrated "dye cushion". After the magnet station, the cartridge was moved to the imaging station within the analyzer and a series of images were acquired in each of the four color channels: red (excitation 635 / 25 nm, emission 680 / 40 nm), yellow (excitation 530 / 20 nm, emission 572 / 23 nm), green (excitation 470 / 40 nm, emission 520 / 40 nm), orange (excitation 569 / 25 nm, emission 609 / 34 nm).

[0224] Imaging of Labeled Cells: The MultiPath analyzer imaging system is a custom-built instrument and software capable of automatically capturing image data from selected wells of a MultiPath cartridge as part of a fully automated test. It uses a custom-designed high-precision 3-axis positioning system to locate each well on a fluorescence-based image acquisition subsystem. The analyzer is capable of imaging in four separate color channels and uses an objective lens, illumination LEDs, a fluorescence filter set, and a camera. The objective lens has a field of view designed to capture an image of the entire cartridge imaging well. The illumination module light source consists of two high-power LEDs per color channel. A series of fluorescent image frames are captured by the camera using a 3.1MP Sony IMX265 monochrome sensor with 12 bits per pixel quantization. A final image for each well is then formed by summing multiple frames. For the red channel, 16 frames were captured with a 100 ms exposure using a 635 / 25 nm excitation and 680 / 40 nm emission filter. For the orange channel, 24 frames were captured with 100 ms exposure using 569 / 25 nm excitation and 609 / 34 nm emission filters. For the yellow channel, 48 frames were captured with 100 ms exposure using 530 / 20 nm excitation and 572 / 23 nm emission filters. For the green channel, 32 frames were captured with 100 ms exposure using 470 / 40 nm excitation and 520 / 40 nm emission filters. The focal plane for imaging the labeled cells was experimentally determined in this example.

[0225] result. FIG. 26 shows a portion of a complete acquired image that detected fluorescence in each of the four color channels, each specific to one of the four input bacteria. Each spot corresponds to a single cell or group of cells. An algorithm is used to identify meaningful objects distinct from artifacts (e.g., debris) and count them as cells. As can be seen in the inset for each bacterium, a similar number of cells was detected as expected, since the input cell concentrations were roughly the same. When overlaid, the spots do not correspond, indicating that with the four different bacterial targets, different objects were observed in each channel, as expected.

[0226] Conclusion: This method allows a single, rapid FISH method to simultaneously detect and quantitate four different bacteria in a single well of a cartridge. Variations:

[0227] This example is illustrative of the multiplexing capabilities of this novel FISH method, and is not limited to the specific details described.Thus, those skilled in the art will easily understand that many variations are possible, including using different probe sequences and nucleic acid structures (PNA, LNA, etc.) and alternative assay chemistries (different detergents, chaotropes, fluorophores, buffers, pH, temperature, reaction time, component concentrations).This methodology can obviously be extended to other biological specimens and other bacterial and non-bacterial pathogens for which specific probes can be designed. Example 6 Automated Rapid AST of E. coli in Clinical Urine Specimens in On-Instrument Cartridges

[0228] Overview: This example demonstrates the use of the inventive system, device, and method to determine antimicrobial susceptibility of targeted bacterial pathogens (in this example, E. coli) in urine in 4 hours without the need for cell purification. The example uses a concerted FISH method for labeling and magnetic sorting, and to quantify specific target cells after differential growth using non-magnified digital imaging. This new method has comparable performance to the gold standard CLSI broth microdilution (BMD) method. Testing Method:

[0229] Urine Specimens: Forty-eight remnant, de-identified urine specimens collected from patients with urinary tract infections (UTIs) and known to contain E. coli were received from Dr. Kirby's laboratory at Beth Israel Hospital (Boston, MA). Samples were received 1-5 days after collection and contained a urine preservative to limit loss of cell viability. For each sample, urine color, pH, and the presence of particulates were recorded. Upon receipt, conventional urine cultures were performed to determine the approximate CFU / mL of bacteria present and to confirm the single or mixed bacterial morphology reported by Dr. Kirby's laboratory. Briefly, a calibrated 1 μL loop was placed into the thoroughly mixed urine sample and 1 μL was spread evenly onto a tryptic soy agar (TSA, BD catalog 221185) plate and incubated in a 35°C incubator for 18-24 hours. The remainder of the urine samples were processed and assayed as described below.

[0230] Urine processing: Prior to testing, urine preservatives and other potentially interfering compounds were removed using size exclusion chromatography. 2.5 mL of each clinically positive urine sample was applied to a pre-washed Zeba™ 7K MWCO spin column (ThermoFisher, Cat. No. 89893) and centrifuged according to the manufacturer's instructions. To investigate bacterial loss after processing, urine cultures were repeated on the treated samples as described above.

[0231] Preparation of magnetic particles: Polyaspartic acid-conjugated magnetic particles (Fluidmag-PAA, Chemicell, catalog 4108) used for nonspecific capture of bacterial cells were dissolved in 50 mM EPPS buffer, pH 8.2 at 2.75 × 10 per mL. 12 Fluorescent magnetic microspheres containing a green dye (Dragon Green Fluorescent Microspheres, BANGS Laboratories, catalog MEDG001) were diluted 1:20 to 3 × 10 particles per mL. 6 The magnetic particles were added to the suspension at a final concentration of 10 ...

[0232] Labeling of bacterial cells at AST time 0: The assay signal at time 0 (T0) before the onset of bacterial growth in the presence or absence of antibiotics was determined for each clinical urine specimen. 30 μL of each treated urine was added to 70 μL of Mueller-Hinton Broth (MHBII) adjusted with 1× cations containing the species-specific Alexa647N-labeled DNA oligonucleotide FISH probe and an unlabeled DNA helper probe. The probe sequences used are shown in Table A. 100 μL of the mixture was then added to wells of a microtiter plate containing dehydrated hybridization buffer (3X SSC (0.45 M NaCl, 0.045 M Na Citrate) buffer (Sigma, Catalog No. S6639), 0.18% Cetrimide (Sigma, Catalog No. H9151), 0.77% CHAPSO (Sigma Catalog No. C3649), 0.72% SB3-12 (Sigma Catalog No. D0431) 0.13 M Guanidine Thiocyanate (Sigma, Catalog No. G9277)). 10 μL of the prepared magnetic particle mixture was then added to the wells. 100 μL of this reaction mixture was transferred to a microtiter plate containing 50 μL of "dye cushion" (50 mM TRIS pH 7.5 (Sigma Cat. T1075), 7.5% v / v Optiprep (Sigma Cat. D1556), 50 mg / mL Direct Black 19 (Orient Cat. 191L)) per well (pre-dried) and incubated for 30 minutes at 35° C. After incubation, the microtiter plate was placed on a magnetic field (Dexter magnetic technologies, Cat. 54170260) for 4 minutes, allowing the fraction containing the magnetic particles, the labeled cells, to migrate through the "dye cushion" near the imaging surface at the bottom of the well.

[0233] Imaging of Labeled Cells: The MultiPath™ Laboratory Imaging System is a custom-built instrument and software capable of automatically capturing image data from selected wells of a microtiter plate. It uses a high-precision linear stage from Prior Scientific (Rockland, MA) to position each well relative to a fluorescence-based image acquisition subsystem. The instrument is capable of imaging in four separate color channels and uses an objective lens, LED illumination, a fluorescence filter set, and a camera. The objective lens has a field of view designed to capture an image of the entire well of the microtiter plate. The illumination module light source consists of two high-power LEDs per color channel. A series of fluorescent image frames are captured by a camera using a 3.1MP Sony IMX265 monochromatic sensor that quantizes 12 bits per pixel. A final image for each well is then formed by summing multiple frames. Sixteen frames were captured with a 100 ms exposure using a 635 / 25 nm excitation filter and a 667 / 30 nm emission filter. Focal particles were imaged with 470 / 40 nm and 520 / 40 nm excitation filters, and two frames were captured with a 20 ms exposure.

[0234] Preparation of antibiotic plates: Microtiter plates were prepared containing six concentrations of each antibiotic in a two-fold serial dilution series, starting at a concentration 10-fold higher than the expected minimum inhibitory concentration (MIC). The antibiotics used were cefazolin, ciprofloxacin, nitrofurantoin, and trimethoprim-sulfamethoxazole. Antibiotic dilutions were verified to be within the appropriate range of tolerability by confirming that the MICs for at least two CLSI QC strains were within the QC range reported in CLSI publication M100Ed29E-2019. The concentrations selected to test each antibiotic straddled the breakpoints reported by CLSI for antibiotics against E. coli. In addition to the wells containing the antimicrobial dilution series, eight wells containing water or diluent were included in the plate to allow for positive and negative growth controls without antibiotic.

[0235] 4 hour growth: While cells were being quantified at time 0, 32.4 μL of processed clinical urine and 75.6 μL of 1.43X MHB II (Teknova, Cat# M5860) were added to each well of the antibiotic plate (which already contained 12 μL of antibiotic). Samples were grown for 4 hours at 35° C. in a standard incubator.

[0236] Labeling of bacterial cells at time 4 hours of growth in AST: After incubating samples for 4 hours (T4) in the presence and absence of antibiotics, cells were labeled and quantified to determine how much growth, if any, had occurred. 100 μL of each well of the incubated sample-antibiotic plate was transferred to the corresponding well of the dehydration buffer plate and combined with the FISH probes, helper probes, magnetic particles, and focusing particles in the same manner as described above for the time 0 assay.

[0237] Comparison Methods: Results for the MulitPath™ assay were compared to broth microdilution (BMD) performed according to CLSI method M07-Ed13E 2018.

[0238] Data analysis and threshold creation: Images captured by the CCD camera were used to estimate the detected cells by an algorithm that takes into account both the number of objects in the field of view and the intensity of those objects. Cell counts based on this detection algorithm were obtained at time 0, as well as at time 4 hours without antibiotic and at time 4 hours with all six concentrations of each antibiotic. For each urine sample per drug concentration, the growth fold was calculated as the signal in the well containing the antibiotic after growth (time 4) relative to the signal in the urine sample before growth (time 0). Using the growth fold and the observation of growth in the corresponding well in the CLSI-compatible broth microdilution, a logistic regression model was used to create a threshold to determine a growth fold cutoff above where cells are growing in the presence of antibiotic (and thus resistant at that concentration) and below where cells are in the process of dying (and thus sensitive at that concentration). The point where the growth fold number is below the determined threshold is the MIC value generated by the assay. Correspondingly, the results were assigned to a category of sensitive, intermediate, or resistant to each antibiotic. All data were then compared to the CLSI standard BMD. Four hours of growth in the absence of antibiotics is the control condition to confirm that viable bacteria are present in the treated urine samples. result.

[0239] Figures 28 to 31 show three examples from our larger data set that demonstrate how this method can be used to obtain MICs for three individual urine samples that match the gold standard broth microdilution method. Figure 28 shows the results of the growth fold number at different antibiotic concentrations for a single clinical urine sample (BIUR0017) for a single drug (nitrofurantoin). The MICs for the broth microdilution match exactly with the MICs determined by the growth fold threshold. Figure 29 shows the results of the growth fold number at different antibiotic concentrations for a single clinical urine sample (BIUR0047) for a single drug (cefazolin). The MICs for the broth microdilution match exactly with the MICs determined by the growth fold threshold. Figure 30 shows the results of the growth fold number at different antibiotic concentrations for a single clinical urine sample (BIUR0057) for a single drug (ciprofloxacin). The MICs for the broth microdilution match exactly with the MICs determined by the growth fold threshold. Figure 31 shows the results of the growth fold number at different antibiotic concentrations for a single clinical urine sample (BIUR0052) for a single drug (trimethoprim / sulfamethoxazole). The MICs for broth microdilution correspond exactly to the MICs determined by growth fold threshold.

[0240] Conclusion: This novel method demonstrates that accurate AST results (MIC determinations) can be achieved by only 4 hours of differential growth of minimally processed urine clinical specimens, notably without lengthy colony purification steps. AST results, whether or not reported as antibiotic susceptibility results in MIC categories, can be conveniently compared to the gold standard broth microdilution method.

[0241] Variations. This example is illustrative of the performance of this novel AST method and is not limited to the specific details contained herein. Those skilled in the art will therefore readily appreciate that many variations are possible, including using different probe sequences and nucleic acid structures (PNA, LNA, etc.), alternative assay chemistries (different detergents, chaotropes, fluorophores, buffers, pH, temperature, reaction times, concentrations of components, etc.), urine concentrations and urine processing procedures. This methodology can also clearly be extended to other antibiotics, biological specimens, and other bacterial and non-bacterial pathogens.

[0242] Example 7 Rapid and accurate antimicrobial susceptibility testing for bacteria in urine samples

[0243] Overview: This example demonstrates the use of the present invention to accurately determine antimicrobial susceptibility of pathogens with known antibiotic susceptibility profiles spiked into bacteria-free urine. Evaluation of differential growth in microbiological media containing antimicrobial agents followed by growth using a FISH method in conjunction with the present invention for quantification of target-specific cells took just 4.5 hours. The new method has comparable performance to the gold standard CLSI broth microdilution (BMD) method. Experimental method.

[0244] Bacterial cell preparation: Fifty bacterial strains with known resistance profiles were taken from either the ATCC or CDC antibiotic resistance banks (AR banks) and are shown in Table A. Bacterial cultures for each of these were obtained by inoculating Trypticase Soy Broth (TSB, Hardy Diagnostics catalog U65) with 3 to 5 colonies from a fresh tryptic soy agar plate (TSA, BD catalog 221185) and growing at 35°C for 1.5 to 3 hours to achieve log phase growth. Optical density at 600 nm was used to estimate cell concentration, and each culture was cultured at approximately 5 x 10 in a cation-adjusted Mueller Hinton II (MHBII, Teknova catalog M5860). 6 Dilutions were made to colony forming units (CFU) / mL.

[0245] Urine Processing: Prior to testing, pooled human urine (Innovative Research, Cat. IRHUURE500ML) was applied to pre-washed Zeba™ 7K MWCO spin columns at a ratio of 4 mL of urine to one pre-washed 10 mL spin column (ThermoFisher, Cat. No. 89893) and centrifuged according to the manufacturer's instructions.

[0246] Preparation of magnetic particles: Polyaspartic acid conjugated magnetic particles (Fluidmag-PAA, Chemicell, catalog 4108) used for non-specific capture of bacterial cells were dissolved in 50 mM EPPS buffer, pH 8.2 at 2.75 × 10 per mL. 12 Fluorescent magnetic microspheres containing a green dye (Dragon Green Fluorescent Microspheres, BANGS Laboratories, catalog MEDG001) were diluted 1:20 to 3 × 10 particles per mL. 6The magnetic particles were added to the suspension at a final concentration of 10 ...

[0247] Labeling of bacterial cells at AST time 0: The assay signal at time 0 (T0), before the onset of bacterial growth, was determined for each bacterium in the presence or absence of antibiotics. 6A reaction mixture was prepared consisting of 10 μL of bacterial dilution at CFU / mL, 60 μL of MHBII (1× final concentration in 100 μL) and the appropriate species-specific Alexa647N-labeled DNA oligonucleotide FISH probe for the target bacterial species and its associated unlabeled DNA helper probe. The probe sequences used are shown in the table in FIG. 36. 100 μL of the mixture was then added to wells of a microtiter plate containing dehydrated hybridization buffer (3× SSC (0.45 M NaCl, 0.045 M Na Citrate) buffer (Sigma, Cat. No. S6639), 0.18% cetrimide (Sigma, Cat. No. H9151), 0.77% CHAPSO (Sigma Cat. No. C3649), 0.72% SB3-12 (Sigma Cat. No. D0431), 0.13 M guanidine thiocyanate (Sigma, Cat. No. G9277)). 10 μL of the prepared magnetic particle mixture was then added to the wells. 100 μL of this reaction mixture was transferred to a microtiter plate containing 50 μL of "dye cushion" (50 mM TRIS pH 7.5 (Sigma Cat. T1075), 7.5% v / v Optiprep (Sigma Cat. D1556), 50 mg / mL Direct Black 19 (Orient Cat. 191L)) per well (previously dried) and incubated at 35° C. for 30 minutes. After incubation, the microtiter plate was placed on a magnetic field (Dexter magnetic technologies, Cat. 54170260) for 4 minutes, allowing the fraction containing the magnetic particles, the labeled cells, to migrate through the "dye cushion" to the vicinity of the imaging surface at the bottom of the well.

[0248] Imaging of Labeled Cells: The MultiPath Laboratory imaging system is a custom-built instrument and software capable of automatically capturing image data from selected wells of a microtiter plate. It uses a high-precision linear stage from Prior Scientific (Rockland, MA) to position each well relative to a fluorescence-based image acquisition subsystem. The instrument is capable of imaging in four separate color channels and uses an objective lens, LED illumination, a fluorescence filter set, and a camera. The objective lens has a field of view designed to capture an image of the entire well of the microtiter plate. The illumination module light source consists of two high-power LEDs per color channel. A series of fluorescent image frames are captured by a camera using a 3.1MP Sony IMX265 monochromatic sensor that quantizes 12 bits per pixel. A final image for each well is then formed by summing multiple frames. Sixteen frames were captured with a 100 ms exposure using a 635 / 25 nm excitation filter and a 667 / 30 nm emission filter. Focal particles were imaged with 470 / 40 nm and 520 / 40 nm excitation filters, and two frames were captured with a 20 ms exposure.

[0249] Preparation of antibiotic plates: Microtiter plates were prepared containing six concentrations of each antibiotic in a two-fold serial dilution series. The two-fold dilution series was prepared at 10-fold higher concentrations than the desired concentration in the final broth microdilution to ensure the correct antibiotic range was obtained with additional cell / urine / media mixtures. 12uL of each antibiotic dilution was then aliquoted into the appropriate wells of a 96-well plate. Different antibodies were tested for different bacteria. Antibiotic dilutions were verified to be within the appropriate range of tolerance by confirming that the MICs for at least two CLSI QC strains were within the QC range reported in CLSI publication M100Ed29E-2019. The concentrations selected to test each antibiotic straddled the breakpoints reported by CLSI for the antibiotic against the appropriate bacterial species, so that category decisions (susceptible / intermediate / resistant) were made from this data. In addition to the wells containing the antimicrobial dilution series, several wells containing water or other dilutions were included for positive growth controls without antibiotics and negative growth (no cells) controls. Antibiotic plates were frozen at -80°C and thawed completely before use.

[0250] 4 hour growth: While the time 0 cells were being quantified, 12 μL of prepared bacterial culture, 36 uL of pooled human urine treated as done for the time 0 assay, 60 uL of 2X MHB II (Teknova, Cat# M5860) and 2 uL of water were added to each well of the prepared antibiotic plate. Samples were grown for 4 hours at 35° C. in a standard incubator.

[0251] Labeling of bacterial cells at time 4 hours of growth in AST: After incubating samples for 4 hours (T4) in the presence and absence of antibiotics, cells were labeled and quantified to determine how much growth, if any, had occurred. 100 μL of each well of the incubated sample-antibiotic plate was transferred to the corresponding well of the dehydration buffer plate and combined with the FISH probes, helper probes, magnetic particles, and focusing particles in the same manner as described above for the time 0 assay.

[0252] Comparison Methods: Results for the MulitPath™ assay were compared to broth microdilution (BMD) performed according to CLSI method M07-Ed13E 2018.

[0253] Data analysis and threshold creation: Images captured by the CCD camera were used to estimate the cells detected by an algorithm that considered both the number of objects in the field of view and the intensity of those objects. Cell counts based on this detection algorithm were obtained at time 0, as well as at time 4 hours without antibiotic and at time 4 hours with all concentrations of each antibiotic. For each bacterial sample per drug concentration, the growth fold was calculated as the signal in the well containing antibiotic after growth (time 4) relative to the signal in the urine sample before growth (time 0). Using the growth fold and the observation of growth in the corresponding well in the CLSI-compatible broth microdilution, a logistic regression model was used to create a threshold to determine a growth fold cutoff above where cells are growing in the presence of antibiotic (and thus resistant at that concentration) and below where cells are in the process of dying (and thus susceptible at that concentration). The point at which the growth fold number falls below the determined threshold is the MIC value generated by the assay. Correspondingly, the results were assigned to a category of susceptible, intermediate, or resistant to each antibiotic. The results were then compared to the MIC values ​​and category calls reported by the ATCC or CDC. The 4 hour growth in the absence of antibiotic is a control condition to ensure that viable bacteria were added to each sample and for use in calculating fold inhibition.

[0254] Additionally, for bacteria tested against ceftazidime (CAZ), the presence of exclusive filamentous bacteria (as easily identifiable visually in FIG. 33, comparing left (normal bacteria) to right (filamentous bacteria)) was taken as an indication of impending cell death at that antibiotic concentration, and the MIC concentration was adjusted accordingly, if possible. In the case of bacteria tested against trimethoprim / sulfamethoxazole (TMP / SXT), a threshold was created based on fold inhibition (assay signal in wells containing bacteria but no antibiotic divided by wells containing both antibiotic and bacteria). result.

[0255] Figure 34 shows how this method can be used to obtain MICs for individual bacteria in the presence of a urine matrix that match MICs published by the CDC or CLSI. This example shows the number of growth folds at different antibiotic concentrations for a single bacterium (K. pneumoniae CDC0126) for a single drug (ciprofloxacin). The published MICs (≧0.25 μg / mL) match exactly with the MICs determined by the novel, rapid AST method described in this invention. The threshold for growth folds (20 in this example) is indicated by a grey horizontal line.

[0256] The table in Figure 35 shows the overall performance across all strains tested. The MICs tested are within essential agreement if the MIC determined by the new AST method is either an exact match or within one two-fold dilution of the published value. All bacteria / antibiotic combinations had 100% essential agreement, except in two cases.

[0257] Conclusion: This novel method shows that determination of MICs consistent with published values ​​for highly characterized bacterial strains with multidrug resistance mechanisms can be made in the context of the sample matrix with only 4 hours of growth.

[0258] Variations. This example is illustrative of the performance of this novel AST method and is not limited to the specific details contained herein. Those skilled in the art will therefore readily appreciate that many variations are possible, including using different probe sequences and nucleic acid structures (PNA, LNA, etc.), alternative assay chemistries (different detergents, chaotropes, fluorophores, buffers, pH, temperature, reaction times, concentrations of components, etc.), urine concentrations and urine processing procedures. This methodology can also obviously be extended to other antibiotics, biological specimens and other bacteria for which specific probes can be designed.

[0259] [Table A-1] [Table A-2] [Table A-3] [Table A-4]

[0260] Example 8 Rapid and accurate automated AST results on clinical urine samples without cell purification

[0261] Overview. This example demonstrates the use of the system and method of the present invention to automatically determine AST results for pathogens in clinical urine samples in 4 hours without the need for lengthy cell purification steps. The automated instrument performs the steps required in cartridges containing reagents to determine antimicrobial susceptibility at a constant physiological temperature. The temperature is compatible with both microbial growth and the method of the present invention for detecting and quantifying target cells. The latter method is performed with the system of the present invention using FISH-based labeling, magnetic sorting, and non-magnified digital imaging.

[0262] The pneumatic subsystem of the instrument is used to automatically dispense the specimen in the cartridge into portions or aliquots containing various antimicrobial agents at different concentrations and microbiological media. One of the portions is used to quantify the pathogen cells prior to growth incubation. The system incubates the cartridge for four hours and then quantifies the number of target cells in the well containing the antimicrobial agent. A comparison of the cell counts in the incubated portion containing the antimicrobial agent to the cell counts measured prior to incubation is used to determine the antimicrobial susceptibility of the pathogen to the various antibiotics.

[0263] This example shows the results of using the automated system, device, and method of the present invention for rapid, automated antimicrobial susceptibility testing directly on clinical specimens from hospitalized patients with E. coli in urine. The present invention achieved accurate performance compared to the gold standard CLSI broth microdilution (BMD) method in just 4 hours. Experimental method.

[0264] Urine Specimens: De-identified urine specimens, remnants collected from patients with urinary tract infections (UTIs) and known to contain E. coli, were received from Dr. Kirby's laboratory at Beth Israel Hospital (Boston, MA). Samples were received 1-5 days after collection and contained a urine preservative to limit loss of cell viability. For each sample, urine color, pH, and the presence of particulates were recorded. Upon receipt, conventional urine cultures were performed to determine the approximate CFU / mL for bacteria present and to confirm the single or mixed bacterial morphology reported by Dr. Kirby's laboratory. Briefly, a calibrated 1 μL loop was placed into the thoroughly mixed urine sample and 1 μL was spread evenly onto a tryptic soy agar (TSA) plate and incubated in a 35°C incubator for 18-24 hours. The remainder of the urine sample was processed and assayed as described below. Preparation of AST Cartridges – Media and Antimicrobials

[0265] A few days prior to preparing the cartridges (see FIG. 37 for a diagram of the cartridge) by dispensing 25 uL of 4X MHB II (Teknova, Cat. No. 101320-356) into each of the eight individual growth wells, growth wells 1 and 2 were for time 0 measurements (see description below) and therefore contained growth medium only. Growth wells 3 and 4 also contained medium only. These wells served as positive controls to ensure that growth was observed over a four hour period. In growth wells 5 and 6, as well as 7 and 8, two concentrations of antibiotic were added. To do this, 4.5 μL of antibiotic concentrated at a factor of 22.2 times higher than the target concentration in micrograms per mL was deposited in the appropriate growth well. The cartridges contained either both ciprofloxacin (CIP) and nitrofurantoin (NIT) or both cefazolin (CFZ) and trimethoprim / sulfamethoxazole (TMP / SXT) in two concentrations. See Figure 38 for the final concentrations of each antibiotic in the cartridge. The media and antibiotics were then dried in a convection oven at 40 °C for 16-20 hours.

[0266] Preparation of AST Cartridges - Hybridization Reagents A hybridization buffer was prepared containing 3X SSC (0.45M NaCl, 0.045M sodium citrate, pH 7.5) (Sigma, Catalog No. S6639), 0.18% w / v cetrimide, 0.77% CHAPSO (Sigma Catalog No. C3649), 0.72% SB3-12 (Sigma Catalog No. D0431), and 0.13M guanidine thiocyanate (Sigma, Catalog No. G9277). Trehalose (Sigma, Catalog No. T9449) was dissolved in this mixture to a final concentration of 10% w / v. This hybridization buffer-trehalose mixture was lyophilized into 8.3 μL volumes of beads. Two 8.3 uL beads were placed into each of the eight reagent wells (see FIG. 37 for location on the cartridge). Preparation of AST Cartridges - Magnetic Particles

[0267] Polyaspartic acid conjugated magnetic particles (Fluidmag-PAA, Chemicell, catalog 4108) were diluted 1:20 in 50 mM Epps buffer, pH 8.2, to a final concentration of 2.75 × 10 per mL containing 10% w / v trehalose (Sigma, catalog no. T9449). 14 To dilute this, fluorescent magnetic microspheres containing a green dye (Dragon Green Fluorescent Microspheres, BANGS Laboratories, catalog MEDG001) were added at a concentration of 3 × 10 particles per mL. 6 The magnetic particle mixture was sonicated for 1 min immediately prior to use to minimize clumping. The mixture was then added to the suspension at a final concentration of 100 × 10 beads (2.64 × 10 per reaction). 12 One magnetic particle lyophilized bead was placed into each of the eight reagent wells along with two hybridization mix beads. Procedure for placing the sample in the cartridge - Urine processing

[0268] Prior to testing, urine preservatives and other potentially interfering compounds were removed using size exclusion chromatography. 2.5 mL of each clinically positive urine sample was applied to a pre-washed Zeba™ 7K MWCO spin column (ThermoFisher, Cat. No. 89893) and centrifuged according to the manufacturer's instructions. Urine cultures were repeated on the treated samples as described above to investigate bacterial loss after processing. Procedure for placing sample in cartridge - placing sample on cartridge

[0269] 750 μL of each processed urine sample was combined with 1705 μL of water and 45 μL of species-specific DNA oligonucleotide fluorescent in situ hybridization (FISH) probe and unlabeled DNA helper probe to create a solution containing 30% v / v final concentration of urine. The oligonucleotides used for each bacterium, their concentrations and dye labels can be found in Figure 39. 1 mL of the mixture was added to the sample pot of the cartridge and the cartridge was placed on the analyzer. Running AST cartridges on an automated analyzer

[0270] The cartridge was then placed on the instrument and all subsequent operations except data analysis were automatic. The urine / water / FISH probe mixture (sample) was first directed under vacuum into the top 8 growth wells of the cartridge. The samples in the first 2 growth wells were then quickly repositioned into the reaction wells to rehydrate the hybridization buffer / FISH probe mix and the lyophilized magnetic particles. The samples were then followed by an imaging window containing 46 μL of dehydrated "dye cushion" (50 mM TRIS pH 7.5 (Teknova, Cat. T5075), 7.5% v / v Optiprep (Sigma, Cat. D1556), 5 mg / mL Direct Black-19 (Orient, Cat. #3222), dried for 3 hours at 60° C. in a convection oven) and incubated for 30 minutes at 35° C. on the analyzer. After this incubation, the cartridge was then relocated to the magnet station and placed over a strong permanent magnet (Dexter magnetic technologies, catalog 54170260) for 4 minutes to allow the bacterial cells interacting with the labeled magnetic particles to migrate closer to the imaging surface. Finally, the cartridge was moved to the imaging station and images were taken using a non-magnifying CCD imager as described below.

[0271] The samples in the remaining six growth wells were kept in place and the bacteria were grown in rehydrated medium with or without antibiotics for 4 hours at 35° C. After growth, the cell suspensions were repositioned into the reagent wells as done for the time 0 assay, and the exact same hybridization reactions, magnetic pulldowns, and imaging were performed as described above. Analyzer imaging system and imaging process

[0272] The MultiPath Analyzer Imaging System is a custom-built instrument and software capable of automatically capturing image data from selected wells of the MultiPath Cartridge as part of a fully automated test. It uses a custom-designed precision 3-axis positioning system to position each well relative to a fluorescence-based image acquisition subsystem. The analyzer is capable of imaging in four separate color channels and uses an objective lens, LED illumination, a fluorescence filter set, and a camera. The objective lens has a field of view designed to capture an image of the entire Cartridge Imaging Well. The illumination module light source consists of two high-power LEDs per color channel. A series of fluorescence image frames are captured by a camera using a 3.1MP Sony IMX265 monochromatic sensor that quantizes 12 bits per pixel. A final image for each well is then formed by summing multiple frames. Sixteen frames were captured with a 100 ms exposure using a 635 / 25 nm excitation filter and a 667 / 30 nm emission filter. Focal particles were imaged with 470 / 40 nm and 520 / 40 nm excitation filters, and two frames were captured with a 20 ms exposure.

[0273] Data Analysis: Images captured by the CCD camera were used to estimate the cells detected by an algorithm that considered both the number of objects in the field of view and the intensity of those objects. Cell counts based on this detection algorithm were obtained at time 0, and at time 4 hours without antibiotic and with both concentrations of each antibiotic. For each urine sample per drug concentration, the fold growth was calculated as the signal in the well containing antibiotic after growth (time 4) relative to the signal in the urine sample before growth (time 0). Comparing the fold growth with the observation of growth in the corresponding well in the CLSI-compatible broth microdilution, a threshold was selected for the growth fold cutoff that maximized agreement with the broth microdilution results. Conditions where cells are growing in the presence of antibiotic (and thus resistant at that concentration) will result in a higher number of growth folds, and conditions where cells are in the process of dying (and thus susceptible at that concentration) will result in a lower number of growth folds. With these cartridges, bacteria in a urine sample are called susceptible if both concentrations of antibiotic show no growth based on their number of growth folds. If there is growth at the lower concentration but not at the higher concentration, the bacteria in the urine sample are intermediate for ciprofloxacin, nitrofurantoin and trimethoprim / sulfamethoxazole, and resistant for cefazolin. If both concentrations of antibiotics show growth based on their growth fold thresholds, the bacteria in the urine sample are called resistant. All susceptible / resistant call data were compared to susceptible / resistant calls made by MIC determination in a CLSI-compliant standard BMD. The 4-hour growth in the absence of antibiotic is the control condition to ensure that viable bacteria are present in the processed urine sample.

[0274] Results. Figure 40 shows the average growth fold of four replicates in two cartridges containing clinical urine sample BIUR0067 that contained an E. coli strain. The graph shows the average growth fold at each of two concentrations of each of ciprofloxacin and nitrofurantoin across four replicates in two different cartridges. Using a growth fold value of 2 for both antibiotics, the MulitPath assay calls both ciprofloxacin (CIP) concentrations as growth and both nitrofurantoin (NIT) concentrations as no growth. Thus, by MulitPath, BIUR0067 is resistant to ciprofloxacin and susceptible to nitrofurantoin. E. coli strains isolated from this urine and tested in CLSI-standard broth microdilution were consistent with these susceptibility / resistance calls.

[0275] Figure 41 shows the average growth fold of four replicates in two cartridges containing clinical urine sample BIUR0084, which contained a K. pneumoniae strain. The graph shows the average growth fold at each of two concentrations of each of cefazolin and trimethoprim / sulfamethoxazole across four replicates in two different cartridges. Using a growth fold value of 2 for both antibiotics, the MulitPath assay calls all of the concentrations of both antibiotics, cefazolin and trimethoprim / sulfamethoxazole, as growth. Therefore, this strain of K. pneumoniae is resistant to both antibiotics. This is consistent with both CLSI-standard broth microdilutions done in-house.

[0276] Conclusion: This example shows the results of using the automated system, device, and method of the present invention for rapid, automated antimicrobial susceptibility testing directly on clinical specimens from hospitalized patients with E. coli in urine. The present invention achieved accurate performance compared to the gold standard CLSI broth microdilution (BMD) method in just 4 hours.

[0277] Variations. This example is illustrative of the performance of this novel AST method on a cartridge and is not limited to the specific details contained herein. Those skilled in the art will therefore readily appreciate that many variations are possible, including using different probe sequences and nucleic acid structures (PNA, LNA, etc.), alternative assay chemistries (different detergents, chaotropes, fluorophores, buffers, pH, temperature, reaction time, concentrations of components), modifications to urine concentration and urine processing procedures and stabilization of reactants and antimicrobial agents, different bacterial targets, different antimicrobial agents, etc. This methodology can also clearly be extended to other biological specimens and other bacterial and non-bacterial pathogens.

[0278] Example 9 Rapid AST methods directly in urine samples are robust to variations in pathogen concentrations

[0279] Overview: Robustness to variable inoculum concentrations is important for rapid AST methods since the concentration of target cells is unknown when testing specimens derived directly from specimens. This example demonstrates the use of the present invention to provide accurate and consistent results when covering a wide range of target cell concentrations versus contrived specimens directly from urine specimens. This example demonstrates that variable cell inputs of E. coli BAA-2469, P. aeruginosa ATCC 27853, K. pneumoniae ATCC 700603 and K. pneumoniae CDC-0043 in the presence of 10% urine achieve accurate AST results compared to broth microdilution (BMD), the gold standard for AST. Experimental procedure.

[0280] Preparation of antibiotic plates: Antibiotic plates containing concentrations of either three to five antibiotics in a two-fold serial dilution series were prepared by dispensing 10 μL of a concentration ten times higher than the desired final concentration into wells of a 96-well plate. The concentrations chosen to test each antibiotic spanned the CLSI-reported MIC for the bacterial strain tested. Plates were prepared with all or a subset of the following antibiotics: cefazolin, ciprofloxacin, levofloxacin, nitrofurantoin, and trimethoprim-sulfamethoxazole. In addition to the wells containing the antimicrobial dilution series, four wells contained water to allow for positive (bacterial growth in the absence of antibiotic) and negative (no bacterial cells) controls.

[0281] Culture preparation: Bacterial cultures for E. coli BAA-2469, P. aeruginosa ATCC 27853, K. pneumoniae ATCC 700603, and K. pneumoniae CDC-0043 were obtained by inoculating Trypticase Soy Broth (TSB, Hardy Diagnostics Cat. U65) with 3 to 5 colonies from a fresh tryptic soy agar plate (TSA, BD Cat. 221185) and growing at 35°C for 1.5 to 3 hours to achieve log phase growth. These cells were cultured at various inocula (2 x 10 3 CFU / mL ~ 1×10 7 The bacteria were diluted in Mueller-Hinton broth (MHBII, Teknova catalog M5860) adjusted with 1x cations to approximately 500 CFU / mL. For more accurate cell concentrations, these estimated bacterial inputs were adjusted using colony counts. Plate counts were determined by diluting log phase cultures to approximately 500 CFU / mL in MHBII, plating 100 μL on TSA plates, and counting colonies after 16 to 24 hours of growth at 35°C. The average plate counts were used to calculate the actual CFU present at each concentration tested.

[0282] Magnetic particle preparation: 2-hydroxypropyltrimethylammonium chloride coated silica magnetic particles (SiMag-Q, Chemicell, catalog 1206-5) were dissolved in 50 mM EPPS buffer, pH 8.2 at 2.75 × 10 per mL. 12 Fluorescent magnetic microspheres containing a green dye (Dragon Green Fluorescent Microspheres, BANGS Laboratories, catalog MEDG001) were diluted 1:20 to 3 × 10 particles per mL. 6 The magnetic particles were added to the suspension at a final concentration of 10 ...

[0283] Labeling of bacterial cells at AST time 0: The assay signal before the onset of bacterial growth (time 0 or T0) was determined for each organism and inoculum in the presence or absence of antibiotics. 10 μL of each sample was added to 80 μL of hybridization buffer, with final concentrations of 3X SSC (0.45 M NaCl, 0.045 M Na citrate, Sigma, Cat. No. S6639), 1% CHAPS (Sigma Cat. No. C3023), 1% NOG (Sigma Cat. No. 08001), Mueller-Hinton Broth adjusted with 1X cations (MHBII), species-specific DNA oligonucleotide FISH probes and unlabeled DNA helper probes. The oligonucleotide probes used are shown in Table B. A final concentration of 10% urine was obtained by adding 10 μL of pooled urine (collected and filtered in-house) directly to the mixture. Then, 10 μL of the magnetic particle mixture prepared as described above was added. 100 μL of this reaction mixture was transferred to a microtiter plate containing 50 μL of "dye cushion" (50 mM TRIS pH 7.5 (Teknova, Cat. T5075), 7.5% v / v Optiprep (Sigma, Cat. D1556), 5 mg / mL Direct Black-19 (Orient, Cat. No. 3222) per well (pre-dried) and incubated at 35° C. for 30 minutes. After incubation, the microtiter plate was placed on a magnetic field (Dexter magnetic technologies, Cat. 54170260) for 4 minutes, allowing the fraction containing the magnetic particles, the labeled cells, to migrate through the "dye cushion" near the imaging surface at the bottom of the well.

[0284] Imaging of Labeled Cells: The MultiPath™ Laboratory Imaging System is a custom-built instrument and software capable of automatically capturing image data from selected wells of a microtiter plate. It uses a high-precision linear stage from Prior Scientific (Rockland, MA) to position each well relative to a fluorescence-based image acquisition subsystem. The instrument is capable of imaging in four separate color channels and uses an objective lens, LED illumination, a fluorescence filter set, and a camera. The objective lens has a field of view designed to capture an image of the entire well of the microtiter plate. The illumination module light source consists of two high-power LEDs per color channel. A series of fluorescent image frames are captured by a camera using a 3.1MP Sony IMX265 monochromatic sensor that quantizes 12 bits per pixel. A final image for each well is then formed by summing multiple frames. Sixteen frames were captured with a 100 ms exposure using a 635 / 25 nm excitation filter and a 667 / 30 nm emission filter. Focal particles were imaged with 470 / 40 nm and 520 / 40 nm excitation filters, and two frames were captured with a 20 ms exposure.

[0285] 4 hour growth: While the cells were being quantified at time 0, 10 μL of each organism inoculum, 10 μL of pooled urine, and 70 μL of 1× MHBII were added to the appropriate wells of an antibiotic plate (already containing 10 μL of antibiotic). Samples were grown for 4 hours at 35° C. in a standard air incubator.

[0286] Labeling of bacterial cells at time 4 hours of growth in AST: After incubating samples with and without antibiotics for 4 hours (T4), cells were labeled and quantified to determine how much growth, if any, had occurred. 10 μL (10%) of the incubated sample-antibiotic plate was transferred to a microtiter plate and combined with 100 μL of hybridization buffer, FISH probe, helper probe, magnetic particles, and focusing particles in the same manner as described above for the time 0 assay.

[0287] Comparison Methods: Results using the novel AST method described herein were compared to broth microdilution (BMD) performed according to CLSI M07-Ed13E 2018.

[0288] Data analysis and threshold creation: Images captured by the CCD camera were used to estimate the detected cells by an algorithm that takes into account both the number of objects in the field of view and the intensity of those objects. Cell counts based on this detection algorithm were obtained at time 0, as well as at time 4 hours without antibiotic and at time 4 hours with all concentrations of each antibiotic. For each sample inoculum per drug concentration, the growth fold was calculated as the signal in the wells containing antibiotic after growth (time 4) relative to the signal in the urine sample before growth (time 0). Using the growth fold and the observation of growth in the corresponding wells in CLSI-compatible broth microdilution, a logistic regression model was used to create a threshold to determine a growth fold cutoff above where cells are growing in the presence of antibiotic (and thus resistant at that concentration) and below where cells are in the process of dying (and thus sensitive at that concentration). The point where the growth fold number is below the determined threshold is the MIC value generated by the assay. Correspondingly, the results were assigned to a category of sensitive, intermediate, or resistant to each antibiotic. All data were then compared to the CLSI standard BMD. Four hours of growth in the absence of antibiotics is the control condition to confirm that viable bacteria are present in the treated urine samples. result.

[0289] Figures 42 to 45 show how this method is robust to changes in inoculum levels while concordant with the gold standard broth microdilution method. Figure 42 compares the results obtained with the new AST method to those of the standard BMD performed at a single concentration for all drugs tested. The third row compares the MICs obtained by the new AST method and the gold standard BMD. All MIC calls were within one two-fold dilution of the CLSI corresponding BMD (essential agreement). The fourth row compares categorical antibiotic susceptibility results (S=susceptible, I=intermediate, R=resistant) based on MIC (categorical agreement). Although concentrations of a subset of Klebsiella give different category calls in broth microdilution than the MIC, all of these were classified as only minor errors by the standard AST methodology. Figure 44 shows the MICs (filled circles) obtained by the novel 4-hour method described above for all inoculum levels against E. coli BAA-2469 compared to the standard broth microdilution method (24-hour BMD, dashed line). All MICs determined by the novel method were within essential agreement (shaded areas). Figure 45 shows the raw data for Figure 43.

[0290] Conclusion: The rapid 4-h AST method presented herein is robust to initial cell concentrations over a wide range of target cell concentrations. Robustness to variable inoculum concentrations is important for rapid AST methods because the concentration of target cells is unknown when testing specimens derived directly from the specimen.

[0291] Variations. This example is illustrative of the performance of this novel AST method and is not limited to the specific details contained herein. Those skilled in the art will therefore readily appreciate that many variations are possible, including using different probe sequences and nucleic acid structures (PNA, LNA, etc.), alternative assay chemistries (different detergents, chaotropes, fluorophores, buffers, pH, temperature, reaction time, concentrations of components), urine concentrations and urine processing procedures. This methodology can also obviously be extended to other biological specimens and other bacterial and non-bacterial pathogens for which specific probes can be designed, as well as to other antimicrobial or chemical agents.

[0292] Example 10 Rapid antimicrobial susceptibility testing for target pathogens in urine clinical specimens containing multiple bacterial species without cell purification

[0293] Overview. Current methods for antimicrobial susceptibility testing require lengthy culture-based colony purification to ensure a pure population of target pathogen cells free of other microorganisms. Colony purification, the usual method, requires 2-5 days to deliver results. During that time, patients are empirically treated with potent broad-spectrum antibiotics that may not be optimal or even completely ineffective at killing the pathogen causing the infection. Additionally, empirical treatment with broad-spectrum antibiotics leads to the spread of antibiotic resistance.

[0294] Current methods require lengthy cell purification processes to determine which antimicrobial agents inhibit the growth of the target pathogen in microbiological media because these methods use non-specific detection methods such as increased turbidity. When using non-specific measurements of cell replicas, one can only know that the growth seen is due to the target pathogen if only cells of the target pathogen are contained. Current antimicrobial susceptibility testing methods must undergo cell purification because most medical specimens are not sterile. The specimens generally contain microorganisms that make up the human microbiome, the benign, normal population of bacteria that live in the human body.

[0295] In contrast, the method of the present invention can achieve accurate antimicrobial susceptibility test results directly from the specimen without a colony purification step. This method differs from current methods in that it evaluates target pathogen-specific growth in a microbiological medium containing an antimicrobial agent.

[0296] This example demonstrates that the rapid antimicrobial susceptibility testing method accurately determines minimum inhibitory concentrations (MICs) for E. coli strains in artificial samples containing urine matrix (10%) for 15 different culture-negative urine samples. Here, we show that antimicrobial susceptibility testing results using the new method are accurate and not significantly affected by off-target bacteria in urine samples containing high concentrations of other microbial species. Experimental procedure.

[0297] Preparation of antibiotic plates: Prior to beginning the experimental procedure, plates containing five concentrations in a two-fold serial dilution series were prepared by dispensing 10 μL of a concentration ten times higher than the desired concentration. The concentrations chosen to test each antibiotic spanned the breakpoints reported by CLSI for antibiotics against E. coli. In addition to the wells containing the antimicrobial dilution series, four wells containing water were included in the plate to allow for positive and negative controls.

[0298] Culture preparation: Three to five colonies of E. coli BAA-2469 and eight other off-target species (S. aureus ATCC 25923, C. freundii ATCC 43864, A. baumannii ATCC 19606, S. epidermidis ATCC 12228, M. luteus (environmental isolate), C. minutissmum ATCC 23348-BAA 949, K. pneumoniae CDC 0043, and K. pneumoniae CDC 0141) were each separately inoculated into 5 mL of Tryptic Soy Broth (TSB, Hardy Diagnostics catalog U65) and incubated at 35°C for 1-2 hours with shaking. The optical density was measured by spectrophotometer and the organisms were diluted in Mueller-Hinton Broth (MHBII, Teknova catalog M5860) adjusted with 1X cations. E. coli was cultured at approximately 5 × 10 6 CFU / mL (final assay concentration is 5 × 10 5 CFU / m), while other off-target species were diluted for various inocula (1 × 10 5 From 5×10 8 CFU / mL).

[0299] Magnetic particle preparation: 2-hydroxypropyltrimethylammonium chloride coated silica magnetic particles (SiMag-Q, Chemicell, catalog 1206-5) were dissolved in 50 mM EPPS buffer, pH 8.2 at 2.75 × 10 per mL. 12 Fluorescent magnetic microspheres containing a green dye (Dragon Green Fluorescent Microspheres, BANGS Laboratories, catalog MEDG001) were diluted 1:20 to 3 × 10 particles per mL. 6The magnetic particles were added to the suspension at a final concentration of 10 ...

[0300] Labeling of bacterial cells at AST time 0: The assay signal before the onset of bacterial growth (time 0 or T0) was determined for each species of E. coli in the presence or absence of antibiotics. 10 μL of each sample was added to 80 μL of hybridization buffer (3X SSC (0.45 M NaCl, 0.045 M sodium citrate) (Sigma, Cat. No. S6639), 1% CHAPS (Sigma Cat. No. C3023), 1% SB3-12 (Sigma Cat. No. 08001), 1X Mueller-Hinton Broth adjusted with cations (MHBII), E. coli-specific DNA oligonucleotide FISH probes and unlabeled DNA helper probes). The probe sequences are shown in the table in Figure 51. A final concentration of 9.1% urine was obtained by adding 10 μL of pooled urine (collected and filtered in-house) directly to the mixture. 10 μL of the magnetic particle mixture prepared as described above was added directly to this mixture. Here, 100 μL of sample containing hybridization mixture, urine, and magnetic particles were transferred to a microtiter plate containing 50 μL of "dye cushion" (50 mM TRIS pH 7.5 (Teknova, Cat. T5075), 7.5% v / v Optiprep (Sigma, Cat. D1556), 5 mg / mL Direct Black-19 (Orient, Cat. No. 3222) per well (previously dried) and incubated at 35° C. for 30 minutes. After incubation, the microtiter plate was placed on a magnetic field (Dexter magnetic technologies, Cat. 54170260) for 4 minutes, allowing the fraction containing the magnetic particles, labeled cells, to migrate through the "dye cushion" near the imaging surface at the bottom of the well.

[0301] Imaging of Labeled Cells: The MultiPath™ Laboratory Imaging System is a custom-built instrument and software capable of automatically capturing image data from selected wells of a microtiter plate. It uses a high-precision linear stage from Prior Scientific (Rockland, MA) to position each well relative to a fluorescence-based image acquisition subsystem. The instrument is capable of imaging in four separate color channels and uses an objective lens, LED illumination, a fluorescence filter set, and a camera. The objective lens has a field of view designed to capture an image of the entire well of the microtiter plate. The illumination module light source consists of two high-power LEDs per color channel. A series of fluorescent image frames are captured by a camera using a 3.1MP Sony IMX265 monochromatic sensor that quantizes 12 bits per pixel. A final image for each well is then formed by summing multiple frames. Sixteen frames were captured with a 100 ms exposure using a 635 / 25 nm excitation filter and a 667 / 30 nm emission filter. Focal particles were imaged with 470 / 40 nm and 520 / 40 nm excitation filters, and two frames were captured with a 20 ms exposure.

[0302] Four hours of growth: E. coli BAA 2469 was tested for their susceptibility to three antimicrobial agents: ciprofloxacin (CIP), levofloxacin (LVX), and nitrofurantoin (NIT) in the presence of Staphylococcus epidermidis, Micrococcus luteus, Corynebacterium minutissimum, Staphylococcus aureus, Acinetobacter baumannii, and Citrobacter freundii. E. coli BAA 2469 was tested against five antimicrobial agents: cefazolin (CFZ), ciprofloxacin (CIP), levofloxacin (LVX), nitrofurantoin (NIT), and trimethoprim-sulfamethoxazole (TMP / SXT) in the presence of Klebsiella pneumoniae. Antibiotic plates containing these antimicrobial agents were prepared according to the method described above. While the cells were quantified at time 0, either E. coli species (5 × 10 6 CFU / mL) 10 μL, off-target species (1 × 10 5 From 5×10 8 10 μL of 100 μL of 1000 CFU / mL, 10 μL of pooled urine, and 60 μL of MHB II (Teknova, Cat# M5860) were added to each well of the antibiotic plate already containing 10 μL of antibiotic. Samples were grown for 4 hours at 35° C. in a static air incubator.

[0303] Labeling of bacterial cells at time 4 hours of growth in AST: After incubating samples for 4 hours (T4) in the presence and absence of antibiotics, cells were labeled and quantified to determine how much growth, if any, had occurred. 10 μL (10%) of the incubated sample-antibiotic plate was transferred to a microtiter plate containing a dried "dye cushion" and combined with 100 μL of the mixture of hybridization buffer, FISH probes, helper probes, magnetic particles, and focusing particles as described above for the time 0 assay.

[0304] Comparison Method: Results from the novel assay method described herein were compared to broth microdilution (BMD) performed according to M07-Ed13E 2018.

[0305] Data analysis and threshold creation: Images captured by the CCD camera were used to estimate the cells detected by an algorithm that takes into account both the number of objects in the field of view and the intensity of those objects. Cell counts based on this detection algorithm were obtained at time 0, as well as at time 4 hours without antibiotic and at time 4 hours with all concentrations of each antibiotic. For each bacterial sample per drug concentration, the growth fold was calculated as the signal in the well containing antibiotic after growth (time 4) relative to the signal in the urine sample before growth (time 0). Using the growth fold and the observation of growth in the corresponding well in CLSI-compatible broth microdilution, a logistic regression model was used to create a threshold to determine a growth fold cutoff above where cells are growing in the presence of antibiotic (and thus resistant at that concentration) and below where cells are in the process of dying (and thus sensitive at that concentration). The point at which the growth fold number is below the determined threshold is the MIC value generated by the assay. Correspondingly, the results were assigned to categories of susceptible, intermediate, or resistant to each antibiotic. result.

[0306] The data shown demonstrates that the 4-hour AST method is robust to non-sterile samples, while the CLSI BMD method with excess bacteria is not. Figure 46 shows data for E. coli BAA-2469 with increasing concentrations of S. aureus ATCC 25923 up to 100-fold excess in the presence of nitrofurantoin. The MIC of E. coli in the CLSI-like broth microdilution method is affected by the addition of S. aureus strains (marked X in the figure), where the MIC increases from 8 in the absence of S. aureus to 32 with 100-fold excess of S. aureus. In contrast, the novel 4-hour AST assay described in this invention (MultiPath, circles) had the same MIC (8) (dashed line) regardless of the amount of S. aureus cells.

[0307] Figures 48-50 show the raw MIC values ​​determined using this novel method (MultiPath) compared to CLSI broth microdilution with only E. coli BAA-2469 present. The table in Figure 47 shows the overall essential agreement for E. coli in the presence of increasing off-target bacteria. Only a single condition - nitrofurantoin with 1e7 Citrobacter freundii - resulted in a lack of essential agreement, but this did not change the categorical susceptible / intermediate / resistant determination, which had 100% agreement across all antibiotics and all off-target bacteria.

[0308] Conclusion: This example demonstrates that using the present invention for antimicrobial susceptibility testing, no cell purification is required to achieve accurate antimicrobial susceptibility test results for the target pathogen, even in samples containing large numbers of other microorganisms of other species.

[0309] Variations: This example is illustrative of the performance of this novel FISH method and is not limited to the specific details contained herein. Those skilled in the art will therefore readily understand that many variations are possible, including using different probe sequences and nucleic acid structures (PNA, LNA, etc.), alternative assay chemistries (different detergents, chaotropes, fluorophores, buffers, pH, temperature, reaction time, concentrations of components), urine concentrations and urine processing procedures. This methodology can also clearly be extended to other biological specimens and other bacterial and non-bacterial pathogens.

[0310] Example 11 Rapid antimicrobial susceptibility testing is accurate for lactam antibiotics in the presence of beta-lactamase-expressing bacteria

[0311] Overview. Current methods for antimicrobial susceptibility testing require lengthy culture-based colony purification to ensure a pure population of target pathogen cells free of other microorganisms. Colony purification, the usual method, requires 2-5 days to deliver results. During that time, patients are empirically treated with potent broad-spectrum antibiotics that may not be optimal or even completely ineffective at killing the pathogen causing the infection. Additionally, empirical treatment with broad-spectrum antibiotics leads to the spread of antibiotic resistance.

[0312] One of the reasons current methods require lengthy cell purification processes to determine which antimicrobial agents inhibit the growth of a target pathogen in a microbiological medium is because they use non-specific detection methods such as increased turbidity. When using non-specific measurements of cell replication, one can only know that the growth seen is due to the target pathogen if only cells of the target pathogen are contained.

[0313] In contrast, the method of the present invention can achieve accurate antimicrobial susceptibility test results directly from specimens without a colony purification step. This method differs from current methods in that it evaluates target pathogen-specific growth in microbiological media containing antimicrobial agents. In another example, the inventors demonstrate that the method of the present invention is accurate in the presence of large numbers of cells from off-target species.

[0314] In this example, we address another challenge that may arise from performing antimicrobial susceptibility testing on target pathogens in the presence of off-target species. Here, we demonstrate that the method of the present invention achieves accurate antimicrobial susceptibility testing results on target pathogens in artificial urine specimens that contain a large number of off-target species that create enzymes known to degrade the antimicrobial agent being tested. In theory, this could change the concentration of the antimicrobial agent significantly enough to alter the results of the antimicrobial susceptibility test.

[0315] In this example, we demonstrate that rapid antimicrobial susceptibility testing achieves accurate antimicrobial susceptibility test results for two carbapenem antibiotics (meropenem and imipenem) in the presence of numerous off-target pathogens that produce enzymes that degrade this type of antimicrobial.

[0316] Experimental Procedures. Preparation of antibiotic plates: Antibiotic plates were prepared as described in the example of the effect of non-sterile samples on target MICs.

[0317] Preparation of cultures: Three to five colonies of E. coli ATCC 25922, a bacterial strain susceptible to most antibiotics, and K. pneumoniae CDC 0141, a strain that expresses the beta-lactamase OXA-181, among many other resistance genes, were separately inoculated into 5 mL of Tryptic Soy Broth (TSB, Hardy Diagnostics catalog U65) and incubated at 35°C for 1-2 hours with shaking. Optical density was measured by spectrophotometer, and organisms were diluted into Mueller-Hinton Broth (MHBII, Teknova catalog M5860) adjusted with 1X cation. E. coli was diluted to 5×10 5 CFU / mL (CLSI standard concentration), while K. pneumoniae was diluted for various inoculations (1 × 10 6 From 5×10 8 CFU / mL).

[0318] Magnetic particle preparation: 2-hydroxypropyltrimethylammonium chloride coated silica magnetic particles (SiMag-Q, Chemicell, catalog 1206-5) were dissolved in 50 mM EPPS buffer, pH 8.2 at 3.75 × 10 per mL. 6 Fluorescent magnetic microspheres containing a green dye (Dragon Green Fluorescent Microspheres, BANGS Laboratories, catalog MEDG001) were diluted 1:20 to 3 × 10 particles per mL. 6 The magnetic particles were added to the suspension at a final concentration of 10 ...

[0319] Labeling of bacterial cells at AST time 0: The assay signal at time 0 (T0) before the onset of bacterial growth in the presence or absence of antibiotics was determined for each clinical urine specimen. 30 μL of each treated urine was added to 70 μL of Mueller-Hinton Broth (MHBII) adjusted with 1× cations containing a species-specific Alexa647N-labeled DNA oligonucleotide FISH probe and an unlabeled DNA helper probe. The probe sequences used are shown in FIG. 54. 100 μL of the mixture was then added to a well of a microtiter plate containing dehydrated hybridization buffer (3X SSC (0.45 M NaCl, 0.045 M Na Citrate) buffer (Sigma, Catalog No. S6639), 0.18% Cetrimide (Sigma, Catalog No. H9151), 0.77% CHAPSO (Sigma Catalog No. C3649), 0.72% SB3-12 (Sigma Catalog No. D0431) 0.13 M Guanidine Thiocyanate (Sigma, Catalog No. G9277)). 10 μL of the prepared magnetic particle mixture was then added to the well. 100 μL of this reaction mixture was transferred to a microtiter plate containing 50 μL of "dye cushion" (50 mM TRIS pH 7.5 (Sigma Cat. T1075), 7.5% v / v Optiprep (Sigma Cat. D1556), 50 mg / mL Direct Black 19 (Orient Cat. 191L)) per well (pre-dried) and incubated for 30 minutes at 35° C. After incubation, the microtiter plate was placed on a magnetic field (Dexter magnetic technologies, Cat. 54170260) for 4 minutes, allowing the fraction containing the magnetic particles, the labeled cells, to migrate through the "dye cushion" near the imaging surface at the bottom of the well.

[0320] Imaging of Labeled Cells The MultiPath™ Laboratory Imaging System is a custom-built instrument and software capable of automatically capturing image data from selected wells of a microtiter plate. It uses a high-precision linear stage from Prior Scientific (Rockland, MA) to position each well relative to a fluorescence-based image acquisition subsystem. The instrument is capable of imaging in four separate color channels and uses an objective lens, LED illumination, a fluorescence filter set, and a camera. The objective lens has a field of view designed to capture an image of the entire well of the microtiter plate. The illumination module light source consists of two high-power LEDs per color channel. A series of fluorescent image frames are captured by a camera using a 3.1MP Sony IMX265 monochromatic sensor that quantizes 12 bits per pixel. A final image for each well is then formed by summing multiple frames. Sixteen frames were captured with a 100 ms exposure using a 635 / 25 nm excitation filter and a 667 / 30 nm emission filter. Focal particles were imaged with 470 / 40 nm and 520 / 40 nm excitation filters, and two frames were captured with a 20 ms exposure.

[0321] Four hours of growth: E. coli was tested for susceptibility to two antimicrobial agents: imipenem and meropenem in the presence of varying K. pneumoniae-OXA inocula. Quantification of cells at time 0 was performed while E. coli species (5 × 10 6 CFU / mL) 10 μL, K. pneumoniae (1 × 10 6 From 1×10 8 10 μL of 1000 CFU / mL or 10 uL of medium (control), 10 μL of pooled urine, and 60 μL of MHB II (Teknova, Cat# M5860) were added to each well of the antibiotic plate already containing 10 μL of antibiotic. Samples were grown for 4 hours at 35°C in a static air incubator.

[0322] Labeling of bacterial cells at time 4 hours of growth in AST: After incubating samples for 4 hours (T4) in the presence and absence of antibiotics, cells were labeled and quantified to determine how much growth, if any, had occurred. 100 μL of each well of the incubated sample-antibiotic plate was transferred to the corresponding well of the dehydration buffer plate and combined with the FISH probes, helper probes, magnetic particles, and focusing particles in the same manner as described above for the time 0 assay.

[0323] Comparison Methods: Results for the MulitPath™ assay were compared to broth microdilution (BMD) performed according to CLSI method M07-Ed13E 2018.

[0324] Data analysis and threshold creation: Images captured by the CCD camera were used to estimate the detected cells by an algorithm that takes into account both the number of objects in the field of view and the intensity of those objects. Cell counts based on this detection algorithm were obtained at time 0, as well as at time 4 hours without antibiotic and at time 4 hours with all six concentrations of each antibiotic. For each urine sample per drug concentration, the growth fold was calculated as the signal in the well containing the antibiotic after growth (time 4) relative to the signal in the urine sample before growth (time 0). Using the growth fold and the observation of growth in the corresponding well in the CLSI-compatible broth microdilution, a logistic regression model was used to create a threshold to determine a growth fold cutoff above where cells are growing in the presence of antibiotic (and thus resistant at that concentration) and below where cells are in the process of dying (and thus sensitive at that concentration). The point where the growth fold number is below the determined threshold is the MIC value generated by the assay. Correspondingly, the results were assigned to a category of sensitive, intermediate, or resistant to each antibiotic. All data were then compared to the CLSI standard BMD. Four hours of growth in the absence of antibiotics is the control condition to confirm that viable bacteria are present in the treated urine samples.

[0325] Results. Figure 52 shows the MICs of E. coli strains susceptible to imipenem in the presence of increasing amounts of K. pneumoniae strains that are resistant to imipenem antibiotics by producing beta-lactamases that degrade the antibiotic. The novel rapid AST method of the present invention is compared to the BMD method. The novel 4.5 hour AST method is not affected by the presence of K. pneumoniae producing even higher concentrations of beta-lactamases that consistently have MICs below 1 μg / mL imipenem. In contrast, the BMD method after 16-24 hours of growth shows an increase in MICs for susceptible E. coli strains with increasing levels of K. pneumoniae, resulting in a false determination of resistance to this antibiotic. Figure 53 shows similar results for the lactam antibiotic meropenem.

[0326] Conclusion: Our novel 4.5 hour AST method demonstrates accurate MIC determination of bacteria susceptible to carbapenem antimicrobial agents, even in the presence of high concentrations of resistant bacteria expressing carbapenemase enzymes that degrade the antibiotics.

[0327] Variations. This example is illustrative of the performance of this novel AST method and is not limited to the specific details contained herein. Those skilled in the art will therefore readily appreciate that many variations are possible, including using different probe sequences and nucleic acid structures (PNA, LNA, etc.), alternative assay chemistries (different detergents, chaotropes, fluorophores, buffers, pH, temperature, reaction times, concentrations of components, etc.), urine concentrations and urine processing procedures. This methodology can also be extended to further pairings of lactam-susceptible bacteria with beta-lactamase-expressing bacteria.

[0328] Example 12 Accurate rapid antimicrobial susceptibility testing of bacteria in urine without culture-based cell purification.

[0329] Summary: Current methods for antimicrobial susceptibility testing require lengthy culture-based colony purification to ensure a pure population of the specimen itself, truly free of pathogen cells. In conclusion, antimicrobial susceptibility test results, which indicate which antibiotic is best at killing the infection-causing pathogen, are not available for 2-5 days. During that time, patients are empirically treated with powerful broad-spectrum antibiotics that may not be optimal at killing the infection-causing pathogen, and may even be completely ineffective. Additionally, empirical treatment with broad-spectrum antibiotics leads to the spread of antibiotic resistance.

[0330] In contrast, the method of the present invention can achieve accurate antimicrobial susceptibility test results directly from specimens without lengthy colony purification steps. Here, we show that the results of the new antimicrobial susceptibility test are not significantly affected when bacteria in urine samples are tested without colony purification. This example demonstrates that the rapid antimicrobial susceptibility test method accurately determines minimum inhibitory concentrations (MICs) for E. coli strains in artificial samples containing urine matrix (10%) for 15 different culture-negative urine samples.

[0331] Experimental Procedure. Urine Specimens: Fifteen culture-negative clinical urine samples (remnants) were purchased from Discovery Life Sciences. Samples were received >7 days after collection and stored at -80°C until use. For each sample, urine color, pH, and the presence of particulates were recorded. Upon receipt, conventional urine cultures were performed on the urine to determine that the sample was culture negative. Briefly, a calibrated 1 uL loop was placed into a thoroughly mixed urine sample, spread evenly on a tryptic soy agar (TSA) plate, and incubated in a 35°C air incubator for 18-24 hours. The remainder of the urine samples were assayed as described below.

[0332] Preparation of antibiotic plates: Microtiter plates were prepared containing six concentrations of each antibiotic in a two-fold serial dilution series, starting at a concentration 10-fold higher than the expected minimum inhibitory concentration (MIC). The antibiotics used were cefazolin, ciprofloxacin, nitrofurantoin, and trimethoprim-sulfamethoxazole. Antibiotic dilutions were verified to be within the appropriate range of tolerability by confirming that the MICs for at least two CLSI QC strains were within the QC range reported in CLSI publication M100Ed29E-2019. The concentrations selected to test each antibiotic straddled the breakpoints reported by CLSI for antibiotics against E. coli. In addition to the wells containing the antimicrobial dilution series, eight wells containing water or diluent were included in the plate to allow for positive and negative growth controls without antibiotic.

[0333] Culture preparation: A logarithmic culture of E. coli (BAA-2469) was grown using 3 to 5 colonies inoculated into 5 mL of Tryptic Soy Broth (TSB, Hardy Diagnostics catalog U65) and incubated with shaking at 35°C for 1-2 hours. Optical density was measured by spectrophotometer and organisms were cultured at 5 × 10 in Mueller-Hinton Broth (MHBII, Teknova catalog M5860) adjusted with 1X cation. 6 CFU / mL (5 × 10 in each 100 μL reaction) 5 The cultures were diluted to a final concentration of CFU / mL.

[0334] Magnetic particle preparation: 2-hydroxypropyltrimethylammonium chloride coated silica magnetic particles (SiMag-Q, Chemicell, catalog 1206-5) were dissolved in 50 mM EPPS buffer, pH 8.2 at 2.75 × 10 per mL. 12Fluorescent magnetic microspheres containing a green dye (Dragon Green Fluorescent Microspheres, BANGS Laboratories, catalog MEDG001) were diluted 1:20 to 3 × 10 particles per mL. 6 The magnetic particles were added to the suspension at a final concentration of 10 ...

[0335] Labeling of bacterial cells at AST time 0: The assay signal before the onset of bacterial growth (time 0 or T0) was determined for each urine sample, in the presence or absence of antibiotics. 10 μL of diluted E. coli was added to 70 μL of hybridization buffer: final concentrations: 3X SSC (0.45 M NaCl, 0.045 M Na citrate) buffer (Sigma, Cat. No. S6639), 1% CHAPS (Sigma, Cat. No. C3023), 1% NOG (Sigma Cat. No. 08001), Mueller-Hinton Broth (MHBII) (from 2X stock) adjusted with 1X cation (Teknova, Cat. No. M5866), as well as non-specific DNA oligonucleotide FISH probes and unlabeled DNA helper probes (see FIG. 58 for probe levels, sequences, and concentrations). A final concentration of 10% urine was obtained by adding 10 μL of each individual's urine directly to the mixture. 10 μL of the magnetic particle mixture prepared as above was added directly to this mixture. Now, 100 μL of sample containing hybridization mixture, urine, and magnetic particles was transferred to a microtiter plate containing 50 μL of "dye cushion" (50 mM TRIS pH 7.5 (Teknova, Cat. T5075), 7.5% v / v Optiprep (Sigma, Cat. D1556), 5 mg / mL Direct Black-19 (Orient, Cat. No. 3222), dried in a convection oven at 60° C. for 3 hours) per well (previously dried) and incubated at 35° C. for 30 minutes. After incubation, the microtiter plate was placed on a magnetic field (Dexter magnetic technologies, Cat. 54170260) for 4 minutes, and the fraction containing the magnetic particles, labeled cells, was moved through the "dye cushion" to the vicinity of the imaging surface at the bottom of the well.

[0336] Imaging of Labeled Cells: The MultiPath™ Laboratory Imaging System is a custom-built instrument and software capable of automatically capturing image data from selected wells of a microtiter plate. It uses a high-precision linear stage from Prior Scientific (Rockland, MA) to position each well relative to a fluorescence-based image acquisition subsystem. The instrument is capable of imaging in four separate color channels and uses an objective lens, LED illumination, a fluorescence filter set, and a camera. The objective lens has a field of view designed to capture an image of the entire well of the microtiter plate. The illumination module light source consists of two high-power LEDs per color channel. A series of fluorescent image frames are captured by a camera using a 3.1MP Sony IMX265 monochromatic sensor that quantizes 12 bits per pixel. A final image for each well is then formed by summing multiple frames. Sixteen frames were captured with a 100 ms exposure using a 635 / 25 nm excitation filter and a 667 / 30 nm emission filter. Focal particles were imaged with 470 / 40 nm and 520 / 40 nm excitation filters, and two frames were captured with a 20 ms exposure.

[0337] 4-hour growth: Spiked culture-negative clinical UTI urine samples were tested for their susceptibility to five antimicrobial agents: cefazolin, ciprofloxacin, levofloxacin, nitrofurantoin, and trimethoprim-sulfamethoxazole. Antibiotic plates containing these antimicrobial agents were prepared according to the method described above. At the same time that the time 0 cell quantification was being done, 10 μL of E. coli, 10 μL of urine, and 70 μL of 1× MHB II (Teknova, catalog M5860) were added to each well of the antibiotic plate. Samples were grown for 4 hours at 35° C. in a static air incubator.

[0338] Labeling of bacterial cells at time 4 hours of growth in AST: After incubating samples for 4 hours (T4) in the presence and absence of antibiotics, cells were labeled and quantified to determine how much growth, if any, had occurred. 100 μL of each well of the incubated sample-antibiotic plate was transferred to the corresponding well of the dehydration buffer plate and combined with the FISH probes, helper probes, magnetic particles, and focusing particles in the same manner as described above for the time 0 assay.

[0339] Comparison Methods: Results for the MulitPath™ assay were compared to broth microdilution (BMD) performed according to CLSI method M07-Ed13E 2018.

[0340] Data analysis and threshold creation: Images captured by the CCD camera were used to estimate the detected cells by an algorithm that takes into account both the number of objects in the field of view and the intensity of those objects. Cell counts based on this detection algorithm were obtained at time 0, as well as at time 4 hours without antibiotic and at time 4 hours with all six concentrations of each antibiotic. For each urine sample per drug concentration, the growth fold was calculated as the signal in the well containing the antibiotic after growth (time 4) relative to the signal in the urine sample before growth (time 0). Using the growth fold and the observation of growth in the corresponding well in the CLSI-compatible broth microdilution, a logistic regression model was used to create a threshold to determine a growth fold cutoff above where cells are growing in the presence of antibiotic (and thus resistant at that concentration) and below where cells are in the process of dying (and thus sensitive at that concentration). The point where the growth fold number is below the determined threshold is the MIC value generated by the assay. Correspondingly, the results were assigned to a category of sensitive, intermediate, or resistant to each antibiotic. All data were then compared to the CLSI standard BMD. Four hours of growth in the absence of antibiotics is the control condition to confirm that viable bacteria are present in the treated urine samples.

[0341] Results. Figures 55 to 57 show that there is little matrix effect on the AST results. Figure 55 shows the MICs for E. coli BAA-2469 determined by the new AST method (black circles) compared to the MICs determined by the gold standard CLSI BMD method in the absence of urine for levofloxacin (dashed line). The shaded areas are the areas of essential agreement, which is generally considered to be within the acceptable error range for a CLSI-compliant BMD process. Most of the MICs for levofloxacin determined for E. coli BAA-2469 using the new AST method were in exact agreement with the CLSI method, with the remaining two falling within a 2-fold essential agreement zone. Figure 56 summarizes the results obtained for all five antibiotics. 100% essential agreement and 100% categorical agreement to the standard BMD was observed across 15 culture-negative clinical urine samples using the new AST method. FIG. 57 shows the MICs determined for 15 culture-negative clinical urine samples spiked with E. coli using the novel AST method compared to the MICs observed in a CLSI-compliant BMD process across the five antibiotics tested.

[0342] FIG. 56 shows 100% essential agreement for levofloxacin with each of the 15 spiked-in culture-negative clinical UTI urine samples versus standard BMD.

[0343] Conclusion: The method of the present invention accurately determined MICs (essentially within the zone of agreement for the gold standard BMD method) for the UTI pathogen (E. coli) for all five antibiotics tested in all 15 separate urine matrices. Thus, this novel 4-hour antimicrobial susceptibility test has the ability to provide accurate results directly from urine specimens without the need for lengthy growth-based colony purification, saving substantial time. Rapid AST results can improve patient care by allowing for prompt initiation of correct and effective antibiotic treatment and avoiding the added spread of antibiotic resistance.

[0344] Variations. This example is illustrative of the performance of this novel AST method and is not limited to the specific details contained herein. Those skilled in the art will therefore readily appreciate that many variations are possible, including using different probe sequences and nucleic acid structures (PNA, LNA, etc.), alternative assay chemistries (different detergents, chaotropes, fluorophores, buffers, pH, temperature, reaction times, concentrations of components) and concentrations of urine. This methodology can also obviously be adapted to other bacterial and non-bacterial pathogens and minimally processed clinical matrices other than urine.

[0345] Example 13 Rapid and accurate polymicrobial multi-target AST

[0346] Overview: Polymicrobial infections are common in many types of infections, including wounds. In such potentially life-threatening infections, it is important to determine which antimicrobial agents may be effective against each infectious agent. Current antimicrobial susceptibility testing methods require 2-5 days to purify each of the numerous infectious agents in a polymicrobial infection before they can be analyzed.

[0347] This example demonstrates the potential of the system and method of the present invention to generate rapid AST results in just 4.5 hours directly from patient samples without the need for lengthy colony purification. The method achieves accurate AST results (MIC values) for each target species in an artificial two-target polymicrobial mixture compared to broth microdilution reference standard results. Experimental procedure.

[0348] Preparation of antibiotic plates: A microtiter plate was prepared containing a two-fold serial dilution series of six ciprofloxacin concentrations. The two-fold dilution series was prepared at 10-fold higher concentrations than the desired concentration in the final broth microdilution to ensure the correct antibiotic range was obtained with additional cell / urine / media mixtures. 10 uL of each antibiotic dilution was then aliquoted into the appropriate wells of a 96-well plate. Antibiotic dilutions were verified to be within the appropriate tolerability range by confirming that the MICs for at least two CLSI QC strains were within the QC range reported in CLSI publication M100Ed29E-2019. In addition to the wells containing the antimicrobial dilution series, sufficient wells containing water or other diluent were included for a positive growth control without antibiotic. Antibiotic plates were frozen at -80°C and thawed completely before use.

[0349] Preparation of Cultures: Both sensitive and resistant strains were selected against four different organisms (E. coli ATCC 25922, E. coli BAA-2469, K. pneumoniae CDC 0076, K. pneumoniae CDC 0043, P. aeruginosa CDC 0233, P. aeruginosa CDC 0236, E. faecalis ATCC 29212, and E. faecium ATCC 19434). The strains tested and their resistance to each antibiotic are shown in Table A. Each strain was grown separately with 3 to 5 colonies inoculated into 5 mL of Tryptic Soy Broth (TSB) and incubated at 35°C for 1 to 2 hours with shaking. The optical density was measured by spectrophotometer, and the organisms were cultured at 1 × 10 in Mueller-Hinton Broth (MHBII, Teknova catalog M5860) adjusted with 1× cation. 7 Diluted to CFU / mL.

[0350] Preparation of magnetic particles: A solution of polyaspartic acid conjugated magnetic particles (Fluidmag-PAA, Chemicell, catalog 4108) was added at 2.75 × 10 per mL in 50 mM EPPS buffer, pH 8.2. 12Fluorescent magnetic microspheres containing a green dye (Dragon Green Fluorescent Microspheres, BANGS Laboratories, catalog MEDG001) were diluted 1:20 to 3 × 10 particles per mL. 6 The magnetic particles were added to the suspension at a final concentration of 10 ...

[0351] Labeling of bacterial cells at AST time 0: The assay signal before the onset of bacterial growth (time 0 or T0) in the presence or absence of antibiotics was determined for each species and strain. 5 μL of target A was added to 5 × 10 per organism. 6For final concentrations of CFU / mL, either 5 μL of Target B or 5 μL of MHB II were combined and added to 80 μL of hybridization buffer (final concentrations: 3× SSC (0.45 M NaCl, 0.045 M sodium citrate pH 7) (Sigma, Cat. No. S6639), 0.25 M guanidine thiocyanate (Sigma, Cat. No. 503-84-0), 5% PEG MW 3350 (Sigma, Cat. No. P-3640), 7.5% Igepal CA-630 (Sigma, Cat. No. I3021), 0.2% Cetrimide (Sigma, Cat. No. H9151), Mueller-Hinton Broth adjusted with 1× cation (MHBII), species-specific DNA oligonucleotide FISH probes, and unlabeled DNA helper probes (sequences and concentrations can be found in Table B). A final urine concentration of 10% was obtained by adding 10 μL of pooled urine (Innovative Research, Cat. IR100007P-24203) directly to the mixture for a total reaction volume of 100 μL. 10 μL of either the SiMag-Q magnetic particle mixture (for conditions in which E. coli, K. pneumoniae and P. aeruginosa strains were labeled) or the Fluidmag-PAA magnetic particle mixture (for conditions in which Enterococcus spp. were labeled) prepared as described above was added directly to this mixture. Here, 100 μL of sample containing hybridization mixture, urine, and magnetic particles was transferred to a microtiter plate containing 50 μL of dye cushion (50 mM TRIS pH 7.5 (Teknova, Cat. T5075), 7.5% v / v Optiprep (Sigma, Cat. D1556), 5 mg / mL Direct Black-19 (Orient, Cat. No. 3222), dried at 60° C.) (Dry-Cushion Plate) and incubated at 35° C. for 30 minutes. After this incubation, the microtiter plate was placed on a strong permanent magnet (Dexter magnetic technologies, Cat. 54170260) for 4 minutes, allowing the bacterial cells interacting with the labeled magnetic particles to migrate to the vicinity of the imaging surface.

[0352] Imaging of Labeled Cells: The MultiPath™ Laboratory Imaging System is a custom-built instrument and software capable of automatically capturing image data from selected wells of a microtiter plate. It uses a high-precision linear stage from Prior Scientific (Rockland, MA) to position each well relative to a fluorescence-based image acquisition subsystem. The instrument is capable of imaging in four separate color channels and uses an objective lens, LED illumination, a fluorescence filter set, and a camera. The objective lens has a field of view designed to capture an image of the entire well of the microtiter plate. The illumination module light source consists of two high-power LEDs per color channel. A series of fluorescent image frames are captured by a camera using a 3.1MP Sony IMX265 monochromatic sensor that quantizes 12 bits per pixel. A final image for each well is then formed by summing multiple frames. Sixteen frames were captured with a 100 ms exposure using a 635 / 25 nm excitation filter and a 667 / 30 nm emission filter. Focal particles were imaged with 470 / 40 nm and 520 / 40 nm excitation filters, and two frames were captured with a 20 ms exposure.

[0353] 4 hours of growth: Polymicrobial samples containing two species were tested for susceptibility to one antimicrobial agent: ciprofloxacin. Antibiotic plates containing these antimicrobial agents were prepared according to the method described above. At the same time that the cells were quantified at time 0, 5 μL of either the labeled or detected species and 5 μL of either bacterial species that may be present in polymicrobial UTI infections (but not labeled) or MHB II as a control, 10 μL of pooled urine, and 70 μL of MHB II were added to each well of the antibiotic plate. Samples were grown for 4 hours at 35° C. in a static air incubator. Each strain in this example served as the labeled target species in one instance and as the unlabeled member of the polymicrobial pair in another instance.

[0354] Labeling of bacterial cells at time 4 hours of growth in AST: After incubating samples with and without antibiotics for 4 hours (T4), cells were labeled and quantified to determine how much growth, if any, had occurred. 10 μL (10%) of the incubated sample-antibiotic plate was transferred to a microtiter plate and combined with 100 μL of hybridization buffer, FISH probe, helper probe, magnetic particles, and focusing particles in the same manner as described above for the time 0 assay.

[0355] Comparison Methods: Results using the novel AST method described herein were compared to broth microdilution (BMD) performed according to CLSI M07-Ed13E 2018.

[0356] Data analysis and threshold creation: Images captured by the CCD camera were used to estimate the detected cells by an algorithm that considers both the number of objects in the field of view and the intensity of those objects. Cell counts based on this detection algorithm were obtained at time 0, as well as at time 4 hours without antibiotic and with all six concentrations of ciprofloxacin. For each sample inoculum per drug concentration, the growth fold was calculated as the signal in the well containing antibiotic after growth (time 4) relative to the signal in the urine sample before growth (time 0). Using the growth fold and the observation of growth in the corresponding well in the CLSI-compatible broth microdilution, a logistic regression model was used to create a threshold to determine a growth fold cutoff above where cells are growing in the presence of antibiotic (and thus resistant at that concentration) and below where cells are in the process of dying (and thus susceptible at that concentration). The point at which the growth fold number lies below the determined threshold is the MIC value generated by the assay. Correspondingly, the MIC results were assigned to a susceptible, intermediate, or resistant category based on the CLSI M100Ed28 2018 guidelines. All data were then compared to the CLSI standard BMD.

[0357] Results. Figure 59, Figure 60 and Figure 61 summarize the results of all 48 different pairwise combinations with the antibiotic ciprofloxacin. Figure 59 shows that all MICs determined for the target bacteria by the new 4.5 hour AST method were within the two-fold tolerance allowed for the gold standard BMD method (determined in the absence of a second bacterium) for each target bacteria, regardless of the presence of a second susceptible or resistant bacterium (referred to as essential agreement). Figures 62 and 63 show that the determination of susceptibility and resistance categories for each target bacteria by the new AST method was not affected by these pairwise combinations and was 100% in agreement with the BMD determinations.

[0358] Conclusion: The AST method of the present invention can accurately determine antibiotic susceptibility for each species in a polymicrobial sample in 4.5 hours without the need for time-consuming colony purification required by current methods. This result demonstrates the potential of the present invention to determine antimicrobial agents that can effectively treat life-threatening polymicrobial infections in a reasonable amount of time rather than the days required by today's methods.

[0359] Variations. This example is illustrative of the performance of this novel AST method and is not limited to the specific details contained herein. Those skilled in the art will therefore readily appreciate that many variations are possible, including using different probe sequences and nucleic acid structures (PNA, LNA, etc.) and alternative assay chemistries (different detergents, chaotropes, fluorophores, buffers, pH, temperature, reaction times, concentrations of components). This methodology can also clearly be extended to other biological specimens, other bacteria, and other antibiotics.

[0360] Example 14 Rapid and accurate detection of multiple target pathogens in a single specimen on a cartridge in an automated instrument

[0361] Overview: Polymicrobial infections, infections caused by two or more bacterial species, are common. Current culture-based and MALDI-TOF-based methods for identifying pathogens require lengthy colony purification steps to separately purify large numbers of cells of each target species. This example demonstrates the use of the FISH method of the present invention to detect and identify multiple target pathogens present in an artificial urine sample in 30 minutes in a disposable consumable cartridge inside an automated analyzer that contains all assay reagents. This example shows the potential of the system and method of the present invention to rapidly and specifically identify multiple target pathogens in polymicrobial infections. Experimental procedure.

[0362] Urine Specimens: Ten culture-negative clinical urine samples (remnants) were purchased from Discovery Life Sciences. Samples were received >7 days after collection and stored at -80°C until use. For each sample, urine color, pH, and the presence of particulates were recorded. Upon receipt, conventional urine cultures were performed on the urine to determine that the sample was culture negative. Briefly, a calibrated 1 uL loop was placed into a thoroughly mixed urine sample, spread evenly on a tryptic soy agar (TSA, BD catalog 221185) plate, and incubated in a 35°C air incubator for 18-24 hours. The remainder of the urine samples were processed and assayed as described below.

[0363] Urine Processing: Prior to performing identification (ID), urine preservatives and other potentially interfering compounds were removed using size exclusion chromatography. 2.5 mL of each clinically negative urine sample was applied to a pre-washed Zeba™ Spin Desalting column, 7K MWCO (ThermoFisher, Cat. No. 89893). Samples were passed through the column by centrifugation as described by the manufacturer.

[0364] Preparation of dehydrated reagents in the cartridge: Prior to performing ID, 45 μL of 2.2× concentrated hybridization buffer (6.7× SSC (1 M NaCl, 0.1 M sodium citrate, (Sigma, Cat. No. S6639), 0.4% w / v cetrimide (Sigma, Cat. No. H9151), 1.71% w / v CHAPSO (Sigma, Cat. No. C3649), 1.6% w / v SB3-12 (Sigma, Cat. No. D0431), and 0.29 M guanidine thiocyanate (Sigma, Cat. No. G9277)) was dispensed into six of the cartridge's reagent wells. Upon rehydration to a final volume of 100 uL after processing through the analyzer, the standard 1× hybridization buffer (3× SSC (0.45 M NaCl, 0.045 M Na citrate), 0.18% cetrimide, 0.77% CHAPSO, 0.72% SB3-12, and 0.13 M guanidine thiocyanate) was realized. 1.8 μL of a mixture of target species-specific DNA oligonucleotide FISH probes and unlabeled DNA helper probes were added to two of the eight reagent wells (N=2 for each target in one cartridge). The E. coli FISH oligonucleotide probe set was added to the reagent wells corresponding to cartridge locations A1 and A2, the K. pneumoniae probe set was added to the reagent wells corresponding to cartridge locations A3 and A4, and the P. aeruginosa probe set was added to the reagent wells corresponding to cartridge locations A5 and A6. These cartridge wells containing hybridization buffer and specific probes were then incubated in a convection oven at 50 °C for 16-20 h to dehydrate the material.

[0365] Magnetic particle preparation: Polyaspartic acid conjugated magnetic particles (Fluidmag-PAA, Chemicell, catalog 4108) were diluted at 2.75 × 10 per mL in 50 mM Epps buffer, pH 8.2, with a final concentration of 10% w / v trehalose (Sigma, catalog no. T9449). 12For this dilution, fluorescent magnetic microspheres containing a green dye (Dragon Green Fluorescent Microspheres, BANGS Laboratories, catalog MEDG001) were diluted 1:20 to a concentration of 3 × 10 particles per mL. 6 The magnetic particle mixture was sonicated for 1 min immediately prior to use to minimize clumping. The mixture was then added to the suspension at a final concentration of 100 × 10 beads (2.64 × 10 per reaction). 12 The beads were lyophilized in 1000 mL of PBS (1000 PAA particles) and one bead was placed into each of the six reagent wells.

[0366] Culture preparation: Logarithmic cultures of three different target pathogens (E. coli ATCC 25922, K. pneumoniae ATCC 13883, and P. aeruginosa ATCC 27853) were grown separately with 3 to 5 colonies inoculated into 5 mL of Tryptic Soy Broth (TSB, Hardy Diagnostics catalog U65) and incubated with shaking at 35°C for 1-2 hours. Optical density was measured by spectrophotometer, and organisms were cultured at approximately 5 × 10 in Mueller-Hinton Broth (MHBII, Teknova catalog M5860) adjusted with 1X cation. 6 Diluted to CFU / mL.

[0367] Labeling and imaging of bacterial cells for identification: Assay signal was determined for each target pathogen in artificial polymicrobial mixtures (total of three combinations of two bacteria) containing the two bacteria of interest at a final concentration of 30% of processed urine. Each polymicrobial combination was tested in 10 unique, different culture-negative clinical samples (total of 30 urines tested). Bacterial target A (approximately 5 × 10 per reaction) was stained with 100% lysine (0.01% lysine) and 100% lysine (0.01% lysine). 5 CFU / mL) 103.5 μL, bacterial target B (approximately 5 × 10 per reaction 5103.5 μL of 10000 CFU / mL, 360 μL of urine, and 633 μL were combined for a total volume of 1.2 mL, and 1 mL of this mixture was transferred to the sample addition port of the cartridge. The cartridge was then placed on the instrument and all subsequent operations were performed automatically. The samples were first directed under vacuum into the six growth wells at the top of the cartridge. The samples were then quickly transferred to the reaction wells to rehydrate the hybridization buffer / FISH probe mix and the lyophilized magnetic particles. The samples were then followed by an optical window containing 45 μL of dehydrated "dye cushion" (50 mM TRIS pH 7.5 (Teknova, catalog T5075), 7.5% v / v Optiprep (Sigma, catalog D1556), 5 mg / mL Direct Black-19 (Orient, catalog #3222), dried for 3 hours at 60 °C in a convection oven) and incubated on the analyzer for 30 minutes at 35 °C. After this incubation, the cartridge was repositioned in the magnet station and placed on a strong permanent magnet (Dexter magnetic technologies, catalog 54170260) for 4 minutes to move the bacterial cells interacting with the labeled magnetic particles to the vicinity of the imaging surface at the bottom of the well. Finally, the cartridge was moved to the imaging station and images were obtained using a non-magnifying CCD imager as described below. Briefly, to image the labeled bacterial cells, each individual well was focused by taking successive images of the fluorescent magnetic microspheres in the green channel, the focal plane determined, and the corresponding images of that location obtained in the red channel.

[0368] Imaging of Labeled Cells: The MultiPath™ Analyzer Imaging System is a custom-built instrument and software capable of automatically capturing image data from selected wells of a MultiPath Cartridge as part of a fully automated test. It uses a custom-designed precision 3-axis positioning system to position each well relative to a fluorescence-based image acquisition subsystem. The analyzer is capable of imaging in four separate color channels and uses an objective lens, LED illumination, a fluorescence filter set, and a camera. The objective lens has a field of view designed to capture an image of the entire Cartridge Imaging Well. The illumination module light source consists of two high-power LEDs per color channel. A series of fluorescence image frames are captured by a camera using a 3.1MP Sony IMX265 monochromatic sensor that quantizes 12 bits per pixel. A final image for each well is then formed by summing multiple frames. Sixteen frames were captured with a 100 ms exposure using a 635 / 25 nm excitation filter and a 667 / 30 nm emission filter. Focal particles were imaged with 470 / 40 nm and 520 / 40 nm excitation filters, and two frames were captured with a 20 ms exposure.

[0369] Data analysis: Images captured by a CCD camera were used to estimate the detected cells by an algorithm that takes into account both the number of objects in the field of view and the intensity of the objects. A channel's signal was considered detected when the assay signal was greater than 130.

[0370] Results. The data demonstrate successful identification of two target pathogens in a single sample without detection of absent pathogens (i.e., no cross-reactivity of the FISH probe to non-target bacteria). Figure 64 shows cartridge runs in which E. coli / K. pneumoniae mixed samples are tested (N=10). Figure 65 shows cartridge runs in which E. coli / P. aeruginosa mixed samples are tested (N=10). Figure 66 shows cartridge runs in which K. pneumoniae / P. aeruginosa mixed samples are tested (N=10). Cartridge number 6 for K. pneumoniae / P. aeruginosa was removed from the analysis since that cartridge was unable to obtain valid results. Additionally, in cartridge number 9 A3 for E. coli / P. aeruginosa, an artifact was observed causing the signal in the well to appear abnormally high, and therefore the duplication of this signal was excluded. The replicate of this omitted point (well A4) did not have this artifact, so K. pneumoniae was still categorized as not detected. The assay signal varied across the different cartridges, but in all cases except those already described, the two bacteria spiked into the culture-negative urine were detected, whereas only a very low signal was observed in the wells containing the probe for the unspiked bacteria.

[0371] Conclusion: This example demonstrates that the isothermal FISH method of the present invention, performed on an automated analyzer containing stabilizing reagents inside a consumable cartridge, can specifically identify multiple target bacterial species in artificial urine samples. This shows the potential of the method to identify multiple pathogens in polymicrobial infections. This example also demonstrates the specificity of the method, since no cross-species detection was observed.

[0372] Variations. This example is illustrative of the performance of this novel FISH method on cartridges and is not limited to the specific details contained herein. Those skilled in the art will therefore readily understand that many variations are possible, including using different probe sequences and nucleic acid structures (PNA, LNA, etc.), alternative assay chemistries (different detergents, chaotropes, fluorophores, buffers, pH, temperature, reaction time, concentrations of components), urine concentrations and urine processing procedures, and modifications to the stabilization of the reactants (lyophilization of components). This methodology can also clearly be extended to other biological specimens and other bacterial and non-bacterial pathogens. Incorporation by Reference

[0373] References and citations to other documents, such as patents, patent applications, patent publications, journals, books, articles, web content, etc. are made throughout this disclosure. All such documents are incorporated herein by reference in their entirety for all purposes. Equivalent

[0374] Various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the entire contents of this document, including references to the scientific and patent literature cited herein. The subject matter herein contains important information, exemplification and guidance that can be employed for the practice of the invention in its various embodiments and equivalents. The present invention provides, for example, the following items. (Item 1) 1. A method for antimicrobial susceptibility testing comprising: obtaining a polymicrobial specimen; dividing the sample into wells, at least some of the wells containing different agents or different concentrations of one or more agents; incubating the specimens in the wells to allow differential growth in response to the different agents or different concentrations of agents; and Counting individual cells of a particular species in each well to identify an agent or concentration of agent that inhibits the growth of said particular species. A method comprising: (Item 2) 2. The method of claim 1, further comprising counting cells in control wells that do not contain an antimicrobial agent after incubation of the control wells. (Item 3) 3. The method of claim 2, further comprising comparing the number of cells counted in each well with the count from the control wells to determine the survival rate of the microorganism when exposed to the different agents or different concentrations of agents. (Item 4) 2. The method of claim 1, wherein the incubating step comprises fluorescently labeling the microorganisms in a species-specific manner, and the counting step comprises imaging each well and counting the fluorescent spots in the image. (Item 5) 5. The method of claim 4, wherein the labeling step uses a nucleic acid or nucleic acid analog probe labeled with a target-specific fluorophore and the counting step utilizes fluorescent in situ hybridization. (Item 6) 2. The method of claim 1, wherein the step does not include nucleic acid amplification. (Item 7) 2. The method of claim 1, wherein the incubating step lasts less than about 1 hour and is performed in a growth medium at a temperature below about 40° C. (Item 8) 2. The method of claim 1, wherein the dividing, incubating and counting steps are carried out using a cartridge containing the wells. (Item 9) transferring a portion of the specimen into the cartridge, the cartridge containing microorganism-binding magnetic beads; and loading the cartridge into an analyzer, the analyzer using a magnet to separate the microorganisms from other portions of the specimen and using an imaging subsystem to perform the counting step; The method of claim 8, further comprising: (Item 10) the analyzer performing the splitting step within the cartridge using a pneumatic subsystem; the wells are in the cartridge and are preloaded with the different drugs or different concentrations of drugs; 10. The method of claim 9, wherein after the dividing and incubating steps, the analyzer transfers the contents of the wells to corresponding reagent wells and exposes the incubated specimens therein to the magnetically coupled magnetic beads and species-specific detectable labels. (Item 11) 10. The method of claim 9, wherein the analyzer uses a magnet to attract the microorganism-binding magnetic beads and bound individual microorganisms to a detection surface within the cartridge. (Item 12) 12. The method of claim 11, wherein the magnet attracts the microorganism-bound magnetic beads through a dye cushion that excludes unbound detectable label from the detection surface. (Item 13) Item 12. The method of item 11, wherein the analyzer uses a carousel and / or a mechanical cartridge transporter to move the cartridge to an imaging subsystem to perform the counting step. (Item 14) 11. The method of claim 10, wherein the species-specific detectable label comprises a fluorescent nucleic acid probe that hybridizes to target nucleic acid of a particular species of microorganism. (Item 15) 15. The method of claim 14, wherein the incubating and counting steps accomplish substantially fluorescent in situ hybridization (FISH) analysis at physiological temperature within the analyzer. (Item 16) 24. The method of claim 23, wherein identifying an antimicrobial agent that inhibits the growth of the microorganism comprises comparing the number of complexes in the incubated sample with the number of complexes in an unincubated sample. (Item 17) 10. The method of claim 9, wherein the analyzer uses the cartridge to perform species-specific enumeration of the individual microorganisms after differential growth. (Item 18) The wells are in a cartridge, and the method comprises: 2. The method of claim 1, comprising the steps of transferring a portion of the sample into the cartridge and loading the cartridge into the analyzer. (Item 19) 20. The method of claim 18, wherein the analyzer manipulates the cartridge to perform the dividing, incubating, and counting steps. (Item 20) 20. The method of claim 18, wherein the sample does not require any chemical or molecular sample preparation techniques by the user prior to the transferring step. (Item 21) 20. The method of claim 18, wherein the transferring step comprises pipetting a portion of the specimen from a collection container into a sample well on the cartridge. (Item 22) 2. The method of claim 1, wherein the specimen is selected from the group consisting of whole blood, positive blood cultures, plasma, serum, urine, sputum, bronchoalveolar lavage fluid, endotracheal aspirate, feces, rectal cerebrospinal fluid, wound, peritoneal fluid, pus, lymph, vaginal secretions, nasal secretions, and body tissue specimens. (Item 23) A method for analyzing microorganisms, comprising: obtaining a specimen containing a microorganism; transferring a portion of said specimen into a well, without any colony or cell purification or culture steps, said well containing a species-specific detectable label and a microorganism-binding magnetic bead; collecting said beads on an imaging surface using a magnet; and imaging said detectable label on said imaging surface, thereby determining the presence of cells of said species in said portion of said specimen. A method comprising: (Item 24) 24. The method of claim 23, wherein the sample does not require any chemical or molecular sample preparation techniques by the user prior to the transferring step. (Item 25) labeling the target microorganisms in said wells with said species-specific detectable label; and Binding microorganisms to the microorganism-binding magnetic beads. 24. The method of claim 23, further comprising: wherein the observing step comprises imaging the labeled, magnet-bound cells without washing away the unbound detectable label. (Item 26) 26. The method of claim 25, further comprising the step of counting individual cells of the labeled magnet-bound cells. (Item 27) 24. The method of claim 23, wherein the wells are provided in a cartridge and the collecting and imaging steps occur while the beads and cells are in the cartridge. (Item 28) 28. The method of claim 27, further comprising the step of loading the cartridge into an analyzer that performs the collecting and imaging steps. (Item 29) 30. The method of claim 28, wherein the analyzer comprises a plurality of subsystems, including at least one imaging subsystem for imaging the labeled microorganisms; and a carousel operable to transport test cartridges between the subsystems. (Item 30) 30. The method of claim 28, wherein the transferring step comprises transferring the specimen into a receiving well of the cartridge, the analyzer splitting the specimen into a plurality of separate wells in the cartridge, and the well is one of the separate wells. (Item 31) 28. The method of claim 27, wherein the species-specific detectable label comprises a fluorescently labeled oligonucleotide probe that specifically hybridizes to nucleic acids of the cells of the species. (Item 32) 32. The method of claim 31, wherein the imaging step comprises fluorescence in situ hybridization (FISH) analysis at a constant physiological temperature within the cartridge. (Item 33) 28. The method of claim 27, wherein collecting the beads on the imaging surface comprises magnetically attracting the beads through the dye cushion in the wells while the dye cushion excludes unbound detectable label from the imaging surface. (Item 34) 24. The method of claim 23, wherein the cells of the species are determined to be present in the specimen within about 30 minutes of the transferring step.

Claims

1. 1. A method for antimicrobial susceptibility testing comprising: dividing the resulting specimen containing a plurality of species of microorganisms into a plurality of wells, the plurality of species of microorganisms including target cells of a bacterial species, the target cells comprising a species-specific sequence on 16S or 23S ribosomal RNA, at least a portion of the plurality of wells being: a) different drugs, the drugs in each well being different from each other, or b) one or more drugs at different concentrations, the concentration of said drugs in each well being different from one another; the steps of: incubating the specimens in the wells to allow differential growth in response to the different agents or different concentrations of one or more agents, wherein the target cells are capable of growing in the absence of the different agents; labeling said target cells with a fluorescently labeled nucleic acid probe, said fluorescently labeled nucleic acid probe binding to said species-specific sequence on 16S or 23S ribosomal RNA; incubating the target cells with magnetic particles, thereby forming complexes of fluorescently labeled target cells bound to magnetic particles, wherein the labeling and incubating the target cells with magnetic particles occur simultaneously; depositing the complex onto a detection surface of an imaging well using magnetic forces; and using non-magnified digital imaging to count individual target cells of said complex in each imaging well to identify an agent or concentration of agent that inhibits growth of said target cells. A method comprising:

2. 10. The method of claim 1, further comprising counting the individual target cells in a control well after incubation of the control well without an antimicrobial agent.

3. 3. The method of claim 2, further comprising comparing the number of individual target cells obtained from the control wells with the number of cells obtained from wells containing growth medium and the antimicrobial agent to identify the different agent or different concentrations of one or more agents that inhibit growth of the target cells.

4. 2. The method of claim 1, wherein said labeling comprises fluorescently labeling said microorganisms and said counting comprises imaging each well and counting fluorescent spots in the image.

5. The method of claim 1 , wherein the dividing, incubating, and counting does not involve nucleic acid amplification.

6. 2. The method of claim 1, wherein incubating the specimen in the well lasts less than 1 hour and is performed in growth medium at a temperature below 40°C.

7. 2. The method of claim 1, wherein said dividing, incubating and counting are performed using a cartridge containing said wells.

8. loading the cartridge into an analyzer, the analyzer using a magnet to separate the target cells from other portions of the specimen, and performing the counting using an imaging subsystem configured to image the individual target cells in the cartridge. The method of claim 7 further comprising:

9. the analyzer performs the splitting step within the cartridge using a pneumatic subsystem configured to drive movement of the analyte within the cartridge by air pressure or vacuum; the wells are in the cartridge and are preloaded with the different drugs or different concentrations of one or more drugs; 9. The method of claim 8, wherein after the dividing and incubating, the analyzer transfers the contents of the well to a reagent well and exposes the incubated specimen therein to target cell-binding magnetic beads and a target cell-specific detectable label.

10. 10. The method of claim 8 or 9, wherein the analyzer uses a magnet to attract complexes of fluorescently labeled target cells bound to magnetic particles to a detection surface in the well within the cartridge.

11. The method of claim 9, wherein the analyzer uses a magnet to attract complexes of fluorescently labeled target cells bound to magnetic particles to a detection surface in the well in the cartridge, the magnet attracting the target cell-binding magnetic beads through a dye cushion that excludes unbound target cell-specific detectable label from the detection surface, the dye cushion containing a dye that absorbs excitation and emission light used by the instrument for imaging.

12. and / or moving the cartridge to an imaging subsystem by rotating a mechanical cartridge transporter; the analyzer using a mechanical cartridge transporter to move the cartridge to an imaging subsystem; The method of claim 10 , wherein the counting is performed and the imaging subsystem is configured to image the individual target cells in the cartridge.

13. 10. The method of claim 9, wherein the incubating, labeling, and counting accomplishes fluorescent in situ hybridization (FISH) analysis at a temperature of 36° C. or greater and less than 40° C. in the analyzer.

14. 9. The method of claim 8, wherein the analyzer uses the cartridge to perform species-specific enumeration of the individual target cells after differential growth.

15. The wells are in a cartridge, and the method comprises:

10. The method of claim 1, comprising the steps of transferring a portion of the specimen into the cartridge and loading the cartridge into an analyzer.

16. 16. The method of claim 15, wherein the analyzer manipulates the cartridge to perform the dividing, incubating, and counting.

17. 16. The method of claim 15, wherein the transferring comprises pipetting a portion of the specimen from a collection container into a well on the cartridge.

18. 2. The method of claim 1, wherein the specimen is selected from the group consisting of whole blood, blood cultures, plasma, serum, urine, sputum, bronchoalveolar lavage fluid, endotracheal aspirate, feces, cerebrospinal fluid, peritoneal fluid, pus, lymph, vaginal secretions, nasal secretions, and body tissue specimens.

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