Analysis instrument

Abstract

To provide a multi-function instrument for automatically and simultaneously carrying out a variety of tests including identification of targets in specimens and antimicrobial susceptibility testing thereof.SOLUTION: Application-specific cartridges are pre-loaded with all required reagents and allow tests to be performed in a single testing device without specimen preparation to, for example, rapidly detect infections, identify infectious pathogens, and analyze their susceptibility to various antimicrobial agents. An instrument includes various stations for carrying out test steps that can be randomly accessed in any order instructed by a computer-controlled instrument.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present disclosure relates to systems and methods for automated testing of analytes. [Background technology]

[0002] Infectious diseases are a leading cause of death and health care costs. Antimicrobial therapy has revolutionized the management of infections and saved countless lives, but overuse of antimicrobial drugs is accelerating the spread of antimicrobial resistance. To ensure that these beneficial drugs are used only when necessary and that the right patients get the right antimicrobial at the right time, faster and more accurate diagnostic methods that can be performed across a wide variety of settings are needed.

[0003] Nosocomial infections are a significant risk to patients and result in severe morbidity and high mortality, as well as significant health care costs. An important factor in the prevalence and severity of nosocomial infections is the occurrence of pathogens that are resistant to multiple antimicrobial agents. Effective treatment of infectious diseases requires rapid diagnosis to detect the infection, identify the infectious agent, and then select the appropriate antimicrobial agent for effective treatment against the agent.

[0004] Traditional methods are time consuming, taking 2-5 days to detect infection, identify pathogens, and determine effective antimicrobial treatment. To protect patients who are in the early stages of infection and in the few days required to determine optimal treatment, physicians initially treat empirically by prescribing potent, broad-spectrum antimicrobial agents. These treatments may be suboptimal or even ineffective. Delays in implementing plausible narrow-spectrum treatments that target a patient's specific infection can have enormous effects on morbidity and mortality. Furthermore, empirical treatments in the absence of diagnostic results or the use of diagnostics prone to false-positive results lead to broad-spectrum administration of potent antimicrobial agents to noninfected patients. Indiscriminate use of antimicrobial agents is a major contributing factor to the spread of target cells with antimicrobial resistance. Finally, delays in implementing optimal treatments result in significant health care costs due to extended hospital stays and costly medical complications. Summary of the Invention [Means for solving the problem]

[0005] Rapid and accurate identification of patients with infectious diseases and rapid implementation of effective treatment for these patients saves lives and reduces the spread of antimicrobial resistance. The present invention provides a system and method that can accurately identify patients with infectious diseases in about 30 minutes and determine targeted treatment for the patient's infection within hours compared to the days required by current methods. Determining effective antimicrobial treatment days earlier can dramatically improve medical outcomes, including preventing deaths.

[0006] To detect infection, the present invention can detect, quantify and identify a wide range of pathogens, including bacteria, fungi, viruses and parasites. The ability of the present invention to detect and quantify diagnostically informative toxins, disease-specific biomarkers, human or host cells, and host-response biomarkers is also beneficial for rapidly and accurately identifying patients with infection. The present invention may include any combination of the above capabilities in a single test to most effectively evaluate patient samples for the presence of infection and determine infectious agents.

[0007] Diagnostically informative host cells include cells indicative of an inflammatory response to infection (e.g., neutrophils), cells infected with a pathogen (e.g., virus-infected cells), or cells indicative of the quality and anatomical origin of a patient specimen (e.g., squamous epithelial cells).

[0008] Examples of toxins that diagnose life-threatening infections include Clostridiodes difficile toxin B, the presence of which indicates C. difficile infection, and Bacillus anthracis lethal toxin (i.e., toxin subunit lethal factor), which indicates anthrax infection, indicative of disease. Host factors that may aid in the identification of infected patients include cytokines, such as IL-4 and IL-6.

[0009] After detecting infection and identifying and quantifying infectious pathogens, the method of the present invention can determine which patient treatments will be most effective. This type of analysis is called antimicrobial susceptibility testing. The present invention differs from current methods for antimicrobial susceptibility testing in that it can obtain accurate results directly from patient specimens in just a few hours, instead of the current conventional methods that take several days. Conventional methods, unlike the method of the present invention, require time-consuming microbiological culture steps to obtain millions of purified pathogen cells. In contrast, the novel antimicrobial susceptibility testing method of the present invention does not require large numbers of cells or cell purification, and can therefore rapidly determine effective treatments directly from patient specimens without time-consuming culture steps.

[0010] The capabilities and utility of the novel potentially medically impactful invention are made possible by the systems and methods of the invention that allow single molecule counting and single cell counting using non-magnified digital imaging of informative biological targets derived directly from patient specimens. Using simple, low-cost cameras, without complex and costly microscopes, and optics for digitally counting microscopic cells and submicroscopic molecules allows for the detection of infections by rapid, highly sensitive, automated quantification of disease-causing toxins and disease-specific biomarkers. The systems and methods of the invention for identifying and digitally counting pathogen cells form the basis for the ability to rapidly determine susceptibility or resistance to antimicrobial agents. In the methods of the invention, whether a pathogen is susceptible to an antimicrobial agent is determined by determining whether the agent stops normal pathogen growth (i.e., an increase in cell number through cell division) when incubated in a microbial nutrient medium. This can be done according to the invention by counting pathogen cells before and after incubation in a medium containing an antimicrobial drug.

[0011] The systems and methods of the present invention allow for the automatic and simultaneous execution of a variety of tests that require only sample input and provide actionable results in a variety of settings ranging from point-of-care to centralized hospitals and reference laboratories. Such testing is made possible by application-specific cartridges preloaded with all necessary reagents, direct sample input into the cartridge, and full automation of all processing and analysis to minimize user hands-on time, benchtop instruments designed for scalable throughput, and instrument combinations.

[0012] The steps of the methods of the invention for counting single molecules and single cells may include fluorescently labeling target molecules or cells, magnetically tagging the targets, depositing the fluorescently labeled magnetic targets onto an imaging surface of a device using magnetic forces, imaging the targets without magnification (or with minimal magnification), and counting the targets using image analysis.

[0013] The present invention allows for the simultaneous processing of the steps outlined above in a cartridge device. A single random access instrument can simultaneously process multiple test cartridges for different diagnostic applications containing different types of patient specimens. The automated nature of the instruments and cartridges of the present invention allows for operation by medical professionals without significant specialized training. Furthermore, the breadth of the potential test menu of the platform for the instrument offers the potential for reducing benchtop space, allowing for greater cost savings in equipment utilization, and enabling diagnostic testing closer to the patient, providing clinicians with potentially life-saving diagnostic information closer to the point of onset when the impact of an infectious disease may be greatest.

[0014] The systems and methods of the present invention use application-specific cartridges that are preloaded with test reagents. The cartridges are preferably assembled and packaged with the necessary test reagents during manufacture and distributed so that the user only needs to add the specimen to be tested (e.g., a respiratory specimen from a patient) and insert the cartridge into the instrument. In some cases, the specimen to be tested, e.g., a blood specimen, may be pre-enriched. For example, blood specimens may undergo pre-enrichment by culture prior to analysis, since many blood infections cannot be directly tested without pre-enrichment due to having too low a concentration of pathogen cells.

[0015] The instruments described herein use a variety of different stations for carrying out the different test steps outlined above. The stations may be positioned around a carousel that is used to receive, store and move application-specific cartridges between stations according to the test being performed. The stations may include a fluidics station for interfacing with the cartridge and manipulating the specimens and reagents therein, a magnetic selection station for magnetically depositing targets on the detection surface of the imaging well of the cartridge, an imaging station for detecting deposited targets in the specimen, and a waste station for disposal of used cartridges. In a preferred embodiment, the test uses a constant temperature throughout all steps or is adjusted so that the interior of the instrument can be maintained at the required temperature and the carousel can serve as a storage station for the incubation steps.

[0016] The instrument can access different stations using a carousel so that the test steps can be performed in the order and timing required for the different types of tests. Precise computer scheduling and computer-controlled access to the different stations in the instrument is used to automatically perform all steps of the different tests without further user input. After loading the cartridge into the instrument, the next interaction of the user can be to receive or view the results of the test, either at the instrument site or remotely. Depending on the test type, the reported results of the platform's automated analysis can indicate the detection of infection; the detection, identification and quantification of pathogens, toxins, biomarkers or diagnostically informative host cells; or antimicrobial susceptibility results and profiles. Some test applications perform different types of measurements on a single specimen in the same cartridge in the same instrument run. In this case, multiple types of results can be reported for a single test.

[0017] The device can read one or more bar codes or other identifiers on the cartridge to associate patient information, test application specific information, or factory information with the cartridge. The device can also receive similar input entered by a user. The device can also use the input to record and track information related to the specimen being tested, including patient information, for reporting results.

[0018] The instrument may include a computer including a processor and non-transitory tangible memory and operable to schedule and manage tests performed within the instrument and to track cartridges within the instrument. The computer may include a user interface for providing and receiving information from a user and displaying results and status information. The computer may be connected to a network and operable to process and send test results over the network to connected devices.

[0019] The instrument of the present invention may include a mechanical conveyor arm for moving the cartridge between the carousel and various stations to perform the required test steps. In a preferred embodiment, the carousel and stations include slots sized to receive and position the cartridge within the station. Rotation of the carousel may align the carousel slot with a corresponding slot in the appropriate station, and the mechanical conveyor arm may be operable to contact a side of the cartridge and slide the cartridge along the aligned slot into the selected station. The mechanical conveyor arm avoids gripping the cartridge, reducing jamming associated with gripping mechanisms. The mechanical conveyor arm may include two rotatable prongs adjacent the cartridge and operable to provide motive force to one side thereof. The sides of the carousel and station slots may provide lateral guidance as the cartridge is slid, avoiding the need for a gripping mechanism to move the cartridge.

[0020] Preloaded cartridges allow manufacturing to control the volume and distribution of reagents, and automated instruments control the execution and timing of test steps. Thus, the system and method can greatly reduce the potential for user error, allowing unskilled staff to perform a variety of tests without specialized training and obtain reliable, actionable results without the delays and costs of dedicated off-site testing.

[0021] In a preferred embodiment, the application-specific cartridge includes a microbe-specific antimicrobial susceptibility testing cartridge for measuring differential growth of pathogens in specimens in the presence of various antimicrobial agents and microbiological growth media selected based on the identity of the pathogen. According to the present invention, patient specimens, such as urine, feces, or blood, are analyzed directly with minimal or no specimen preparation or culture. Specimens processed according to the present invention are identified and exposed to various antimicrobials or other treatment modalities, allowing the selection of the most effective treatment. Microbial infections can be identified and appropriate treatments determined in just a few hours, greatly reducing delays in appropriately targeted treatment and avoiding the need for empirical treatment with aggressive broad-spectrum antimicrobials. The present invention allows health care providers to prescribe effective treatments initially to properly treat infected patients. Thus, the present invention provides an opportunity to improve patient outcomes and reduce the spread of antimicrobial resistance.

[0022] Detection of infection, identification of targets, and determination of effective treatments are achieved directly from patient specimens such as urine, sputum or other respiratory specimens, blood, stool, wound specimens, or cerebrospinal fluid with little or no specimen preparation steps. For example, urine specimens are pipetted directly into the test device for pathogen identification (ID) and antimicrobial susceptibility testing (AST) that can be completed in a few hours. This is in contrast to current culture-based methods that require one or more days of colony purification to produce a large population of pure microbial cultures for testing. The present invention provides test devices and instruments that can accept and process patient specimens internally to identify microorganisms or cells and / or determine therapeutic susceptibility and efficacy, all within the test device. Multiple target cells or pathogens in the specimen can be identified and susceptibility to multiple antimicrobial drugs or treatments can be tested in a single device. The test systems and methods of the present invention are robust with respect to specimen matrix, variable inoculum, and the presence of commensal microorganisms in the specimen. The tests of the present invention also provide accurate results for polymicrobial infections.

[0023] The system and method of the present invention allows specimen to be directly processed and imaged to determine the presence and identity of target cells present in specimen.As described above, the processing and imaging steps are performed with specimen in a test device such as a cartridge, and little or no specimen preparation is required outside the test device.By eliminating time-consuming specimen preparation techniques and using target-specific distinguishable labels, the system and method of the present invention allows targets in specimen to be identified and enumerated in just 30 minutes or less.

[0024] The systems and methods of the present invention can be used to identify pathogens causing infectious diseases. For example, the methods of the present invention include a novel method for identifying and quantifying pathogens directly in patient specimens without the need for culture-based microbiological pre-enrichment or nucleic acid amplification. In the method, the target pathogen(s) in a single reaction mixture are enumerated by labeling using a combination of fluorescent in situ hybridization (FISH)-based methods and magnetic selection that can be performed in about 30 minutes in a microtiter plate or cartridge in the device described herein.

[0025] The systems and methods of the present invention can be used for diagnostic antimicrobial susceptibility testing (AST), i.e., to determine which antimicrobials are capable of preventing the growth of a microbial pathogen in a patient specimen. This information provides a clinician with information about which antimicrobial should be used to effectively treat that particular patient's infection.

[0026] Antimicrobial susceptibility testing can be thought of as a stepwise 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.

[0027] 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.

[0028] Although both the conventional and inventive methods for antimicrobial susceptibility testing follow the steps described above, the inventive method determines the antimicrobial susceptibility profile of a pathogen in a matter of hours, whereas the conventional method requires several days. The rapid antimicrobial susceptibility test results using the inventive method result 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.

[0029] 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.

[0030] Several novel aspects of the systems and methods of the present invention provide the ability to rapidly deliver antimicrobial susceptibility results directly from patient specimens.

[0031] First, patient specimens generally contain orders of magnitude fewer cells than are required for conventional antimicrobial susceptibility testing. In contrast to current methods that rely on culture pre-enrichment, the method of the present invention can enumerate small numbers of pathogen cells by highly sensitive single-cell counting using non-magnified digital imaging. Furthermore, because the method enumerates small numbers of individual cells, it is possible to very quickly - within just a few generations of bacteria - determine whether cells have increased in number in an aliquot containing antimicrobial drug and growth medium.

[0032] Second, patient specimens contain a sample matrix and commensal microorganisms unrelated to infectious pathogens. Guidelines for conventional methods (e.g., from the Clinical Laboratories Standards Institute or the European Committee on Antimicrobial Susceptibility Testing) 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 are free of sample matrix.

[0033] 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.

[0034] 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.

[0035] In contrast to conventional methods and others that use non-specific methods to detect whether a pathogen grows in the presence of an antimicrobial drug, requiring purified pathogen cells, the methods of the present invention use pathogen-specific detection to assess the growth of a pathogen in an antimicrobial drug. Because only disease-causing pathogen cells are enumerated after the incubation step (and not any commensal microorganisms), the methods of the present invention can be used to directly determine antimicrobial susceptibility in non-sterile primary specimens containing one or many commensal microbial species.

[0036] The system and method of the present invention can be used to identify target cells or microorganisms and test the antimicrobial susceptibility of targets in specimens separately or in the same device. In a preferred embodiment of the present invention, the test device divides the specimen internally into separate parts, where some of these parts can be incubated in the presence of different antimicrobial agents before imaging to determine differential growth. One or more of these parts can be directly processed and imaged to provide a baseline reference for determining growth, growth inhibition or morphological changes in the incubated parts. By quantifying the growth of the parts incubated in different antimicrobial agents, the effectiveness of each antimicrobial agent in reducing or preventing the growth of the target can be determined. By observing changes in target cell count or cell morphology, the effectiveness of treatment can be determined.

[0037] The test devices and instruments described herein allow for the identification and testing of the effectiveness of drugs against various classes of targets (e.g., viruses, human cells, bacterial cells, or fungal cells), and also allow such identification and antimicrobial susceptibility testing to be performed simultaneously on a single device against many different targets, thereby enabling the development of a single instrument to perform tests typically performed by multiple test devices designed for different testing applications (e.g., blood, urinary tract, gastrointestinal, and respiratory infections). Thus, the instruments and test devices described herein provide robust functionality.

[0038] Once a target is identified, antimicrobial susceptibility testing may include antimicrobial agents or treatments appropriate for that target (e.g., those commonly used in treatments or known to inhibit the growth of the identified target). As previously mentioned, identification of the target cell or microorganism may be important to determine the appropriate target-specific treatment. Identification may be performed using the same processing and imaging techniques used in the antimicrobial susceptibility testing methods described herein, and may be performed using the same types of devices, instruments, and methods used for differential growth or treatment efficacy analysis. In certain embodiments, identification and treatment susceptibility testing may be performed on the same specimen (split into separate portions) in the same device. Target identification may also be performed using other techniques not included in the systems and methods of the present invention, such as, for example, amplification with target-specific primers, immunoassays, mass spectrometry, nucleic acid sequencing, or oligonucleotide probe array analysis. When identification is performed separately, antimicrobial susceptibility testing devices containing appropriate reagents and antimicrobials for the pathogen to be identified may be used in accordance with the present invention.

[0039] Detecting differential growth in the presence of various antimicrobial agents may require different amounts of time depending on the target pathogen, but is significantly reduced from the number of days required for standard antimicrobial susceptibility testing techniques.For example, differential growth of microorganisms commonly associated with urinary tract infections can be observed in urine specimens using the techniques of the present invention after about 4 hours or less.As described above, identifying and quantifying microorganisms in specimens can be achieved in 30 minutes or less, thereby obtaining antimicrobial susceptibility test results within a few hours after introducing the specimen into the test device.

[0040] In certain embodiments, a test suite of a consumable cartridge device can be used for identification (ID) and antimicrobial susceptibility testing (AST) for symptomatic infections (e.g., pneumonia or urinary tract infections). Such a test suite may be referred to as an ID / AST test and may include an ID cartridge and a symptomatic infection-specific family of AST cartridges, each containing a valid antimicrobial for testing an individual pathogen or group of related pathogens.

[0041] The ID cartridge in such a symptomatic infectious disease testing suite can detect infection, identify the infectious pathogen(s) in the specimen, and quantify the bioburden or concentration of the infectious pathogen(s) in the specimen. The same cartridge can also simultaneously test specimens for diagnostically informative markers including host response biomarkers (e.g., cytokines) and inflammatory cells (e.g., neutrophils), or cellular markers of sample quality (e.g., squamous cells) on the same device. The ability to combine detection and quantification of pathogens, biomarkers, and diagnostically informative host cells in the same specimen, cartridge, and instrument is a novel and potentially powerful advantage of the systems and methods of the present invention.

[0042] If an infection is detected and a pathogen is identified using an ID cartridge (or an alternative identification method separate from the method of the present invention), an AST cartridge is selected from a family of AST cartridges for AST analysis. The AST cartridge selected for analysis contains an appropriate antimicrobial drug that can be used to treat the particular pathogen, and an appropriate FISH reagent for enumerating the particular pathogen.

[0043] The antimicrobial susceptibility test results obtained using the systems and methods of the present invention are then used to determine an antimicrobial susceptibility profile for the infectious pathogen and to inform patient treatment decisions so that patients can be treated with effective antimicrobial agents.

[0044] Detectable labels may include target-specific fluorescent oligonucleotide probes (including probes containing modified nucleotides or nucleotide analogs) that bind to targets or fluorescent antibodies, specific and non-specific ligands, lectins, dyes, and pigments. In certain embodiments, magnetic tags are used in combination with detectable labels that bind to target microorganisms before magnetically selecting and imaging the target microorganisms. Separation may occur within the test device described herein, and a magnetic field may be used to deposit the labeled microorganisms onto a detection surface in the test device that is imaged. In certain embodiments, a dye cushion layer, as described in U.S. Pat. No. 9,643,180, which is incorporated herein by reference, may be used in the separation and imaging steps to minimize or eliminate sample preparation steps by the user, to eliminate washing steps, and to reduce background signals. Digital non-magnified imaging techniques, as described in U.S. Pat. No. 9,643,180 and U.S. Pat. No. 8,021,848, each of which is incorporated herein by reference, may be used to quantify labeled microorganisms, including single cells, for example.

[0045] An aspect of the present invention provides a method for determining antimicrobial susceptibility. Such a method preferably includes obtaining a specimen from a patient suspected of having a symptomatic infection, the specimen type potentially containing an infectious pathogen(s). The specimen is then introduced into a test device and divided into multiple aliquots. One aliquot is immediately analyzed to determine a baseline concentration of pathogen cells before incubation. For this aliquot, the pathogen cells are fluorescently labeled, magnetically tagged, attracted through a dye cushion, deposited on the imaging surface of an imaging well in the cartridge, imaged, and quantified using image analysis. The other aliquots are incubated at 35° C. in the presence of growth medium and various antimicrobial agents, all within the test device. After differential growth in the presence of various antimicrobial agents, the pathogen cells are fluorescently labeled, magnetically tagged, attracted through a dye cushion, deposited on the imaging surface of an imaging well in the cartridge, imaged, and quantified using image analysis. The number of pathogen cells enumerated in the aliquot containing the antimicrobial is compared to the number of pathogen cells enumerated initially (before incubation) to determine the antimicrobial susceptibility profile and which antimicrobial will be effective to treat the patient.

[0046] The method of the present invention may include the steps of detecting infection, and detecting and identifying infectious pathogen cells before introducing the specimen into the test device, and selecting a plurality of different antimicrobial agents based on the identity of the target cells or microorganisms. Identifying the target pathogen preferably includes exposing a first specimen from a patient to a magnetic tag and a fluorescent label that can bind to a first target, so that a complex containing the magnetically tagged and fluorescently labeled target is specifically formed. A magnetic field is applied to the test device to attract the complex to a detection surface; the detection surface is imaged to detect and quantify the detectable label, where the presence and concentration of the detectable label indicates whether and how much the target pathogen is present. The detection step may take less than about 30 minutes.

[0047] For antimicrobial susceptibility testing applications of the present invention, target pathogen cells can be detected after differential growth as described above.

[0048] In the systems and methods of the present invention, a similar strategy can be used to detect and quantify intracellular targets (e.g., toxins, biomarkers, host response factors, virus-specific molecules, or virus particles). Target-specific magnetic tags and fluorescent labels are preferably used to bind to such targets to form complexes. The systems and methods of the present invention are used to deposit these complexes on imaging surfaces for imaging and enumeration by image analysis. Target-specific magnetic tags and fluorescent labels for detecting intracellular targets are preferably magnetic and fluorescent particles conjugated to target-specific binding agents (e.g., antibodies, aptamers, receptors, ligands). The specific formation of magnetically tagged and fluorescently labeled target complexes occurs in a variety of ways. Either the magnetic tag or the fluorescent label can be designed to specifically bind to the target. Alternatively, both the magnetic tag and the fluorescent label can be designed to specifically bind to the target. In either case, magnetic selection combined with imaging of the magnetically selected complex results in the detection and enumeration of specific targets in the specimen. There are various mechanisms by which the magnetic tag and the fluorescent label can be associated with the target.

[0049] There are various ways in which magnetic tags or fluorescent labels can bind to targets nonspecifically. For example, binding magnetic tags or fluorescent labels that bind to conserved sites across targets of various categories can be achieved by conjugating the magnetic tags or fluorescent labels to moieties that bind to the sites (e.g., antibodies or other protein binding partners, lectins, or ligands). Magnetic tags or fluorescent labels can also bind nonspecifically due to common chemical or colloidal properties. For example, positively charged magnetic particles or fluorescent labels can bind nonspecifically to bacterial cells, which are generally negatively charged. Cells can be nonspecifically labeled with various dyes (e.g., calcofluor) or fluorogenic dyes (e.g., propidium iodide, fluorescein diacetate). Dyed fluorescent particles can be used as fluorescent labels that bind nonspecifically to target cells by virtue of their chemical or colloidal properties, or by conjugating them to nonspecific binding molecules such as those mentioned above.

[0050] Magnetic tags or fluorescent labels can also be selected in various ways so that they specifically bind to targets. For example, magnetic tags (or fluorophores) can be conjugated to antibodies that bind to target-specific antigens. For a similar effect, magnetic tags or fluorophores can be conjugated to molecules (e.g., avidin) that specifically bind to ligands (e.g., biotin) that are bound (or can bind) to such target-specific antibodies. Cells can also be specifically labeled by reassociation or hybridization with target-specific nucleic acid probes (or nucleic acid analog probes) that are themselves labeled with fluorophores. Dyed fluorescent particles can be used as fluorescent labels that specifically bind to target cells by conjugating them to target-specific binding molecules such as those described for certain embodiments, and the labeling and imaging steps include exposing the analyte moiety to fluorophore-labeled target-specific binding molecules and magnetic particles, which bind to the target to form complexes; applying a magnetic field to the test device to attract the complexes to the detection surface; and imaging the detection surface. The fluorophore-labeled target-specific binding molecules may include oligonucleotide probes that specifically bind to the target cells. The exposing and imaging steps may include fluorescent in situ hybridization (FISH) methodology and analysis.

[0051] The method of the present invention may include a step of determining a recommended antimicrobial agent for the patient, which usually includes an antimicrobial agent or other treatment determined to inhibit the growth of the target. The determination of the treatment to inhibit the growth of the target can be performed about 4 hours after the specimen is introduced into the test device. The body specimen may be a tissue specimen (e.g., a wound or biopsy specimen) or a body fluid specimen. Preferred body fluids for use with the present invention include, but are not limited to, respiratory (e.g., sputum endotracheal aspirate, protected specimen brush, bronchoalveolar lavage), blood, urine, feces, swabs (e.g., nasal, oral / pharyngeal, surgical site, skin and soft tissue, rectal) and cerebrospinal fluid.

[0052] In a particular embodiment, the present invention provides a system for determining the therapeutic susceptibility of target cells or microorganisms in a specimen. A preferred system includes a test device and an instrument or instruments that can be operated to receive a specimen containing a body fluid from a patient and a target cell or microorganism. The instrument is preferably operable to: manipulate the test device to divide the specimen into a plurality of portions in the test device; incubate these portions in the presence of a plurality of different therapeutic agents in the test device; fluorescently label and magnetically tag the target cells in the portions incubated in the test device; separate the magnetically tagged and fluorescently labeled target complexes from unbound fluorescent labels; image these portions in the test device and quantify the target complexes to determine which treatment(s) inhibit the replication of the target cells.

[0053] The system may include a test device that can be used to detect infections and identify pathogens for microbiological applications. A patient sample may be added to the device, where it may be divided into multiple aliquots, each of which may be contacted with a magnetic tag and multiple types of target-specific detectable labels (e.g., binding molecules with distinct fluorophore labels) such that labeled, magnetically tagged target complexes are formed; a magnetic field is applied to the test device to attract the complexes to a detection surface; the detection surface is imaged to detect the labeled complexes, and detection of a complex labeled with a particular detectable label in a particular aliquot indicates the presence of a particular target.

[0054] The instrument may be operable to contact specimen aliquots with various antimicrobial agents for antimicrobial susceptibility applications; contact aliquots or portions containing the specimen with magnetic tags and detectable labels selected such that a complex involving the target, magnetic tag, and detectable label is specifically formed; apply a magnetic field to the test device to attract the complex to a detection surface; image the detection surface; and perform image analysis to determine a test result.

[0055] Detecting the number of identified target cells or microorganisms in each of the incubated samples may include contacting the incubated samples with a magnetic tag and a detectable label selected to specifically form a complex between the target, the magnetic tag and the detectable label; applying a magnetic field to the complex to attract the complex to a detection surface; and imaging the detection surface to determine the effect of each of the therapeutic agents on the growth of each of the identified targets.

[0056] The multiple aliquots may be combined with the same antimicrobial agent present at different concentrations, preferably corresponding to two-fold serial dilutions of the antimicrobial agent or to concentrations corresponding to CLSI breakpoints for antimicrobial susceptibility testing. The number of such portions and the concentrations of the antimicrobial agent may be selected to obtain a susceptibility / resistant result, a categorical (susceptible, intermediate resistant, resistant or SIR result) or a minimum inhibitory concentration (MIC) result. Appropriate concentrations of antimicrobial agents for specific microbial pathogen species are described by the Clinical Laboratory Standards Institute (CLSI). [Brief description of the drawings]

[0057] [Figure 1] FIG. 1 is a schematic diagram of the present invention.

[0058] [Diagram 2] FIG. 2 shows non-magnified digital imaging of 500 nm fluorescent particles.

[0059] [Diagram 3] FIG. 3 is a schematic diagram of an exemplary method of the present invention.

[0060] [Figure 4] FIG. 4 shows background rejection with a dye cushion.

[0061] [Diagram 5]FIG. 5 is a diagram of steps of an exemplary method of the present invention.

[0062] [Figure 6] FIG. 6 illustrates an exemplary test cartridge.

[0063] [Figure 7] FIG. 7 is a perspective view of the cartridge.

[0064] [Figure 8] FIG. 8 illustrates a workflow for performing antimicrobial susceptibility testing of specimens containing bacteria.

[0065] [Figure 9] FIG. 9 is a diagram illustrating an instrument useful in the systems and methods of the present invention.

[0066] [Figure 10-1] FIG. 10A is a top view of a carousel within the device.

[0067] [Figure 10-2] FIG. 10B is a perspective view of the carousel.

[0068] [Figure 11] FIG. 11 is a diagram showing an exemplary computer and device configuration.

[0069] [Figure 12] 12A and 12B are diagrams illustrating an exemplary mechanical conveyor arm.

[0070] [Figure 13] FIG. 13 is a photograph showing an exemplary device.

[0071] [Figure 14] FIG. 14 is a photograph showing an exemplary cartridge loaded with a specimen.

[0072] [Figure 15] FIG. 15 is a photograph showing an exemplary cartridge being loaded into an exemplary instrument.

[0073] [Figure 16] FIG. 16 is a photograph of a carousel in an exemplary instrument with cartridges loaded.

[0074] [Figure 17] FIG. 17 is a side view of the internal components of a device according to one embodiment of the present invention.

[0075] [Figure 18] FIG. 18 is a side view of a device according to an embodiment of the present invention.

[0076] [Figure 19] FIG. 19 is a front view of a device according to one embodiment of the present invention.

[0077] [Figure 20] FIG. 20 is a side view of a device according to an embodiment of the present invention.

[0078] [Figure 21] FIG. 21 is a rear view of a device according to one embodiment of the present invention.

[0079] [Figure 22] FIG. 22 illustrates one embodiment of a top view of the device layout.

[0080] [Diagram 23] FIG. 23 illustrates an embodiment of a carousel.

[0081] [Figure 24] FIG. 24 illustrates an embodiment of a displacement arm.

[0082] [Diagram 25] FIG. 25 illustrates an embodiment of a movement arm.

[0083] [Figure 26] FIG. 26 illustrates an embodiment of an imaging module.

[0084] [Figure 27] FIG. 27 is a top view of the imaging module.

[0085] [Figure 28] FIG. 28 illustrates an embodiment of an optical assembly.

[0086] [Figure 29] FIG. 29 illustrates an embodiment of a fluidic module.

[0087] [Diagram 30] FIG. 30 illustrates an embodiment of a fluidic module.

[0088] [Diagram 31] FIG. 31 illustrates a top view of an embodiment of a magnetic module.

[0089] [Diagram 32] FIG. 32 illustrates a side view of an embodiment of a magnetic module.

[0090] [Diagram 33] FIG. 33 is a schematic diagram of the method of the present invention in an imaging well, with drainage of the specimen preparation due to a dye cushion and washing steps, all steps being performed in a cartridge on an automated instrument without user intervention.

[0091] [Diagram 34] FIG. 34 illustrates an embodiment of a Catheter Associated Urinary Tract Infection (CAUTI) ID test according to the methods of the present invention.

[0092] [Diagram 35]FIG. 35 illustrates an embodiment of a CAUTI AST test according to the methods of the present invention.

[0093] [Diagram 36] FIG. 36 is a graph showing sensitive FISH detection of E. coli in urine.

[0094] [Figure 37] FIG. 37 is a graph showing the inclusiveness of the FISH assay for E. coli.

[0095] [Figure 38] FIG. 38 is a graph showing the specificity of the FISH technique for E. coli.

[0096] [Figure 39] FIG. 39 is a table showing CAUTI ID testing according to the methods of the present invention.

[0097] [Diagram 40] Figure 40 is a graph showing a comparison of MICs between the CAUTI AST test and the overnight broth microdilution reference method. Dark shading indicates an exact match and light shading indicates a difference of 1x2-fold dilution.

[0098] [Diagram 41] FIG. 41 is a graph showing the limit of detection (LoD) for E. coli.

[0099] [Diagram 42] FIG. 42 is a graph showing the limit of detection (LoD) of P. aeruginosa.

[0100] [Diagram 43] FIG. 43 is a graph showing the limit of detection (LoD) for K. pneumoniae.

[0101] [Diagram 44] FIG. 44 is a table of the probe sequences used in this example.

[0102] [Diagram 45] FIG. 45 is a graph showing the average signal (n=3) plotted for 11 E. coli strains.

[0103] [Diagram 46] FIG. 46 is a graph showing the percentage of input cells detected (determined by plate count).

[0104] [Figure 47] FIG. 47 is a table showing inclusion results for four additional bacterial species.

[0105] [Figure 48] FIG. 48 is a table showing the probe sequences used in this example.

[0106] [Figure 49] Figure 49 is a table showing the bacterial species and strains tested.

[0107] [Figure 49] FIG. 49 is a table showing challenge bacteria for testing specificity of E. coli detection.

[0108] [Figure 50] FIG. 50 is a table showing the probe sequences used in this example.

[0109] [Figure 51] FIG. 51 is a graph showing the specific detection of E. coli and the absence of detection of eight challenge bacteria.

[0110] [Figure 52] FIG. 52 is a graph showing the specific detection of E. coli and the absence of detection of eight additional challenge bacteria.

[0111] [Figure 53]FIG. 53 is a portion of the entire image captured.

[0112] [Figure 54] FIG. 54 is a table of the probe sequences used in Example 4.

[0113] [Figure 55] Figure 55 is a graph showing BIUR0017 with nitrofurantoin.

[0114] [Figure 56] FIG. 56 is a graph showing BIUR047 with cefazolin.

[0115] [Figure 57] FIG. 57 is a graph showing BIUR057 with ciprofloxacin.

[0116] [Figure 58] FIG. 58 is a graph showing BIUR052 with trimethoprim / sulfamethoxazole.

[0117] [Figure 59] FIG. 59 is a table of the probe sequences used in this Example 6.

[0118] [Figure 60] Figure 60 is a diagram showing how this method can be used to generate a MIC.

[0119] [Figure 62] Figure 62 shows overall performance across all strains tested.

[0120] [Figure 60] FIG. 60 is a photograph showing a visual comparison of normal bacteria (left panel) and filamentous fungi (right panel).

[0121] [Figure 61]FIG. 61 is a graph showing that the MIC generated by the novel rapid AST method described in this invention is called at 0.25 μg / mL.

[0122] [Figure 62] Figure 62 is a table of AST results.

[0123] [Figure 63] FIG. 63 is a table of the probe sequences used in Example 7.

[0124] [Figure 64] FIG. 64 is a photograph showing the Multipath™ UTI-AST cartridge.

[0125] [Figure 65] FIG. 65 is a table showing the antibiotic concentrations tested.

[0126] [Figure 66] FIG. 66 is a table of the oligonucleotides used in Example 8.

[0127] [Figure 67] Figure 67 is a graph showing the results for BIUR0067.

[0128] [Figure 68] Figure 68 is a graph showing the results for BIUR0084.

[0129] [Figure 69] FIG. 69 is a table comparing the results obtained.

[0130] [Figure 70] FIG. 70 is a graph showing MIC results for various inoculum levels generated using the new methods described herein compared to the conventional BMD method.

[0131] [Figure 71]FIG. 71 is a table that was generated showing a summary of the MIC results for the various inoculum levels.

[0132] [Figure 72] FIG. 72 is a table of the probe sequences used in Example 9.

[0133] [Figure 73] Figure 73 is a graph showing data for E. coli BAA-2469 in the presence of nitrofurantoin.

[0134] [Figure 74] Figure 74 is a table showing the overall intrinsic concordance of E. coli in the presence of increasing off-target bacteria.

[0135] [Figure 74] Figure 74 is a table showing a summary of the concordance between E. coli and standard BMD for off-target microorganisms at various inoculum levels.

[0136] [Figure 75] FIG. 75 is a table showing the agreement with standard BMD for E. coli and off-target microorganisms (S. aureus, Staphylococcus epidermidis, and Citrobacter freundii) at various inoculum levels.

[0137] [Figure 76] FIG. 76 is a table showing the agreement with standard BMD for E. coli and off-target microorganisms (Micrococcus luteus, Acinetobacter baumannii, Corynebacterium minutissimum) at various inoculum levels.

[0138] [Figure 77] FIG. 77 is a table showing agreement with standard BMD for E. coli and an off-target microorganism (K. pneumoniae) at various inoculum levels.

[0139] [Figure 78] FIG. 78 is a table of the probe sequences used in this Example 10.

[0140] [Figure 79] FIG. 79 is a graph showing the MICs of imipenem-susceptible E. coli strains in the presence of increasing amounts of K. pneumoniae strains resistant to imipenem antibiotics.

[0141] [Figure 80] FIG. 80 is a graph showing similar results for the lactam antibiotic meropenem.

[0142] [Figure 79] Figure 79 shows a comparison between a new rapid AST method and the BMD method.

[0143] [Figure 80] Figure 80 shows that the E. coli MICs were consistent with various inoculations of carbapenem-hydrolyzing B-lactamase-producing resistant strains of K. pneumoniae using the above method, but not with standard BMD.

[0144] [Figure 81] FIG. 81 is a table of the probe sequences used in Example 11.

[0145] [Figure 82] FIG. 82 is a graph showing the essential agreement across 15 urine samples.

[0146] [Figure 83] FIG. 83 shows 100% intrinsic agreement and 100% categorical agreement with standard BMD for each of the 15 spiked culture-negative clinical UTI urine samples.

[0147] [Figure 84]Figure 84 shows the MICs for 15 E. coli spiked urine samples determined by the new AST method compared to the standard BMD method ("CLSI Compliant"). Concentrations are in micrograms / ml.

[0148] [Figure 85] FIG. 85 is a table of the probe sequences used in this Example 12.

[0149] [Figure 86] FIG. 86 is a photograph showing that S. aureus cells (left panel) are detected as bright fluorescent spots, whereas the medium without cells (right panel) contains only objects categorized as debris.

[0150] [Figure 87] FIG. 87 is a photograph showing S. aureus cells (left panel) and TSB medium only (right panel).

[0151] [Figure 88] Figure 88 is a graph showing the results of the MultiPath assay.

[0152] [Figure 89] FIG. 89 is a table showing the ciprofloxacin-susceptible and -resistant strains used in this example.

[0153] [Figure 90] FIG. 90 is the first half of a table of the probe sequences used in this Example 13.

[0154] [Figure 91] FIG. 91 is the second half of a table of the probe sequences used in this Example 13.

[0155] [Figure 92] FIG. 92 is a table showing the essential concordance for polymicrobial infections with two target organisms.

[0156] [Figure 93] FIG. 93 is a table showing category agreement for polymicrobial infections.

[0157] [Figure 94] FIG. 94 is a graph showing cartridge runs testing E. coli / K. pneumoniae mixed samples (N=10).

[0158] [Figure 95] FIG. 95 is a graph showing cartridge runs testing E. coli / P. aeruginosa mixed samples (N=10).

[0159] [Figure 96] FIG. 96 is a graph showing cartridges tested with K. pneumoniae / P. aeruginosa mixed samples.

[0160] [Figure 97] FIG. 97 is a table, "Table A of Example 14," which shows that the target pathogens were detected while other non-target pathogens were not detected.

[0161] [Figure 98] FIG. 98 is a table, “Table B of Example 14,” showing the probe sequences used in Example 14.

[0162] [Figure 99] FIG. 99 is a graph showing that the method returned to the predicted detection limit. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0163] The present invention provides a system and method for performing different tests simultaneously and automatically using a single instrument. Tests intended to be performed by the instrument include identifying target cells, molecules, viruses, or microorganisms in a specimen and performing antimicrobial susceptibility testing analysis with little or no specimen preparation by the user. The present invention provides a system and method for detecting, quantifying, and identifying target pathogens, diagnostically informative host cells, and diagnostically informative molecular biomarkers in a specimen. In embodiments of the present invention, different types of assays or evaluations are performed on a single analyte. In some embodiments of the present invention, a set of tests or assays are performed on a single specimen in a single cartridge. Assays according to the present invention detect specific molecular and cellular targets and determine effective treatments.

[0164] Target microorganisms, pathogens, and cells intended to be tested using the systems and methods of the present invention include viruses, bacterial or fungal cells, human cells (including but not limited to white blood cells, squamous epithelial cells, or human cells infected with viruses), or other eukaryotic cells (including parasites, animal, and plant cells). Target molecules intended to be tested include target-specific antibodies, nucleic acid probes, ligands, aptamers, and statins. Target cells, molecules, viruses, or microorganisms in a specimen can be analyzed for differential growth in the presence of various antimicrobial agents, or the effect of a therapeutic agent on cell viability, morphology, viral load, cell functionality, or other measures of therapeutic efficacy can be determined using the systems and methods described herein.

[0165] For analysis of urine for common causes of urinary tract infections, specimen manipulation, incubation, processing, and analysis steps are performed in a single test device in less than about 30 minutes for target cell, molecule, virus, or microorganism identification, and less than about 4.5 hours for antimicrobial susceptibility testing. Analysis times can vary based on the target being analyzed (e.g., depending on the growth rate of the target) and based on the therapeutic agent being tested. The systems and methods detect specific targets using non-magnified digital imaging and image analysis to accurately and immediately quantify the target in the specimen.

[0166] The embodiments of the present invention allow testing to be performed with minimal or no sample preparation by the user. By reducing external steps and using imaging methods and image analysis that can quantify single cells or colony forming units (CFUs) in certain cases, actionable results directly from patient samples for target cell, molecule, virus, or microorganism identification and antimicrobial susceptibility testing can be obtained in hours, compared to days with traditional techniques. Variations that may be required across different target cells, molecules, viruses, and microorganisms can include target-specific binding molecules (e.g., target-specific antibodies, nucleic acid probes, ligands, aptamers, and statins), treatments tested (e.g., antivirals, antimicrobials, or other treatments), incubation times, features analyzed, image analysis algorithms, parameters, and results reported.

[0167] The testing device can be automatically manipulated by an instrument to perform each of the steps of dividing the specimen, culturing in the presence of different antimicrobial agents or other treatments, and processing and imaging the resulting specimen portions to measure differential growth and determine the effectiveness of the antimicrobial agents tested.

[0168] The application-specific cartridge may include a series of interconnected wells preloaded with the necessary reagents for a particular test. The user need only add the sample to the cartridge for the desired test and sample type and then load the cartridge into the instrument for automated processing. The instrument may scan the label containing both information about the application-specific cartridge itself and the patient sample information. Alternatively, the operator may input the information through a user interface. The instruments described herein may include stations for performing various steps that may be required for different tests, and may include a carousel or other mechanism for storing multiple in-process cartridges and moving them between stations as needed for performing the test steps.

[0169] The cartridge may include wells as well as various valves and channels for connecting different wells therein as needed to perform the steps of the desired test. For example, a cartridge for antimicrobial susceptibility testing may include a series of separated wells preloaded with growth medium and various antimicrobials to be analyzed. The cartridge may also include wells for processing the specimens after growth and labeling targets therein, as well as imaging wells that provide a detection surface for imaging the labeled targets. The instrument may include a fluidic module that interfaces with the cartridge to allow external manipulation of the valves and to apply pressure to the cartridge as needed for various tests to connect different wells and move specimen volumes between them.

[0170] The instrument can be an automated benchtop instrument designed to accommodate a menu of application-specific consumable cartridges for major infectious diseases. Due to unique non-magnified digital imaging technology, the instrument allows for high performance. The instrument allows for elimination of user steps, use of inexpensive reagents, and streamlined instrument use, resulting in the advantages of low cost and ease of use.

[0171] The instrument achieves single molecule counting and counts individual cells without magnification. The instrument is unique for its ability to rapidly and sensitively detect target toxin molecules, biomarkers, cells, and antimicrobial resistance. Figure 1 shows how the imaging technique of the instrument detects individual target molecules tagged with fluorescent nanoparticles without the use of magnification. Illuminating a molecule tagged with a fluorescent particle causes the labeled target to emit a photon. The photon strikes a CMOS chip in a digital camera (such as in a cell phone) that contains an array of independent light-sensitive pixel elements. Thus, the pixel element that overlies an individual target is "illuminated" as a white spot in the resulting image (Figure 2, showing unmagnified digital imaging of a 500 nm fluorescent particle). A computer instantly enumerates the illuminated pixels, indicating the number of targets present. At low analyte concentrations, digitally counting the individually labeled targets results in a better signal-to-noise ratio compared to the more common method of integrating the signal over a detection area. Non-magnified imaging allows for real-time imaging of a large field of view - enabling rapid detection of small numbers of targets in large volumes of specimens. An important technical advantage arising from the method's innovative non-magnified digital imaging approach is that the technique can detect very low levels of molecules or cells rapidly and with very low-cost components.

[0172] The unique dye cushion layer allows the instrument technique to rapidly and specifically count target molecules in complex samples without sample preparation (Figure 3). Figure 3 shows a schematic diagram of the imaging well method of the present invention. The dye cushion eliminates the need for sample preparation and washing steps. All steps are performed in the cartridge running on the automated instrument without user intervention. The liquid sample potentially containing the target is added to the clear bottom imaging well, which contains two types of dry reagents: dye (e.g. Direct Black) and density agent (OptiPrep). The dry dye cushion reagent on the bottom surface of the imaging well forms a novel high density layer when it contains water. The target-specific fluorescent and magnetic nanoparticles are stabilized as small lyophilized spheres (approximately 1 mm in diameter).

[0173] In one example, Clostridium difficile is the target molecule. Magnetic nanoparticles are coated with antibodies specific for one antigenic site on the C.difficile toxin molecule, and fluorescent nanoparticles are coated with complementary antibodies that bind to a distinct antigenic site on the same molecule. When the dry reagent is hydrated with the analyte, two layers are formed: a high-density dye cushion layer and an assay layer. In the assay layer, the target molecule binds to the magnetic and fluorescent nanoparticles, tethering them together. The high concentration (approximately 109 / ml) and small size (200-500 nm) of the particles drive rapid binding kinetics with only diffusive mixing, thereby simplifying instrumentation by eliminating the need for mechanical mixing functionality.

[0174] By placing the imaging well on a permanent magnet for 3 minutes, the magnetic particles - and any fluorescent particles that are connected to them via target molecules - are attracted through the dye cushion layer and deposited on the imaging bottom surface. Imaging and counting of the captured fluorescent particles is performed in real time using non-magnified digital imaging as described above. Since a single molecule can connect a fluorescent particle to a magnetic particle at low target concentrations, counting the number of magnetically deposited particles corresponds to the number of captured target molecules, and this is done without the use of magnification.

[0175] The dye cushion eliminates sample preparation and washing steps. Figure 4 shows that the dye cushion completely blocks the intense fluorescence of tens of millions of highly fluorescent unbound particles. In one particular embodiment, the dye cushion is composed of two inexpensive organic chemicals: Direct Black (a common dye in the coating industry) and OptiPrep (an iodized density agent used in cell separation). The dye cushion layer passively forms a densely colored layer that absorbs both the excitation and emission wavelengths of light. This layer prevents light from reaching the unbound fluorescent particles in the assay layer (which are present in the millions and are otherwise very bright). Similarly, the dye cushion optically and physically isolates the analyte from the imaging surface, making the assay robust to even the most challenging analyte matrices without the need for user sample preparation. Thus, the dye cushion innovation eliminates the need for user sample preparation and all assay washing steps, significantly reducing the cost and complexity of instrumentation.

[0176] The present invention can be used to detect a wide range of analytes. In addition to detecting molecular targets such as toxins and biomarkers, the instrument can also detect and count cellular pathogens (e.g., bacteria, fungi, and parasites), viruses, and diagnostically important human cells. For example, the technology can detect infections and identify bacterial pathogens in as little as 30 minutes. In addition, antimicrobial susceptibility testing allows targeted antimicrobial therapy to be selected to treat an infection in as little as four hours, compared to two to four days using traditional culture testing.

[0177] 5 illustrates steps of an exemplary method 101 of the present invention. A patient sample 104 can be obtained 103. The sample 104 can include a bodily sample from a patient, such as blood or a portion thereof (e.g., plasma or serum), urine, saliva, sputum, cerebrospinal fluid, amniotic fluid, stool, peritoneal fluid, pus, lymph, vaginal secretions, vomit, sweat, or any other fluid obtained from the human body. The sample can include tissue and other non-fluid samples, for example, biopsies and swab samples, such as nasal, rectal, vaginal, surgical site, skin, mucosal tissue, and oral swab samples. In certain embodiments, non-medical samples can be tested, including, but not limited to, veterinary samples, environmental samples, agricultural samples, and food samples.

[0178] In some embodiments, the specimen 104 can be directly introduced 107 into the testing device 109, generally without any pre-processing steps. For example, a urine specimen directly from a patient can be pipetted into the testing device 104 without the need for prior specimen preparation, including mixing with reagents, target purification, processing to remove components of the specimen, centrifugation, biochemical enrichment, colony purification, or other processing. Once the specimen 104 is obtained 103, it is introduced 107 into the testing device 109.

[0179] In some embodiments, the step includes specimen processing 106. A patient sample 104 can be obtained 103. The patient sample 104 can then be subjected to specimen processing 106 before being introduced 107 to a testing device 109. In certain embodiments, the specimen processing can be pre-specimen processing. Some embodiments can include on-board specimen processing including filtration, cell purification, specific depletion, enrichment for specific classes of cells or analytes, or any combination thereof.

[0180] 6 illustrates a test cartridge 201 for use in various systems and methods of the present invention. The test cartridge 201 includes an inlet 203 for receiving a specimen, a dividing well 205, a reagent well 209, an imaging well 211, and a channel 213 for moving the specimen between the wells, and a valve 207 for controlling that movement. The cartridge 201, when interfaced with a control device, is operable to receive the specimen at the inlet 203 and to divide the specimen into a portion through the channel 213 and into the dividing well 205.

[0181] The cartridge 201 can interface with a fluidic module of an instrument described herein, for example, at air port 217. The valve 207 can control the movement of fluid between the divided wells 205 and the processing 209 and imaging wells 211 by opening and closing the connection therebetween. The valve 207 allows one or more portions of the specimen to bypass the divided wells 205 and proceed directly to the processing 209 and imaging wells 211 to provide an unincubated reference (n t0) can be provided. Valve 207 can be of a sliding bar 223 design as shown in Figure 6. The sliding bar 223 can be manipulated in coordination with pressure or vacuum applied to air port 217 by the fluidic module of the instrument described herein such that the computer controlled instrument times and directs fluid movement within the cartridge according to the planned parameters of the cartridge-specific test being performed.

[0182] The divided wells 205 can be pre-filled with growth medium and / or one or more antimicrobial agents. Valves 207 allow portions of the specimen to be held in the divided wells 205 while being incubated in the presence of different antimicrobial agents for any period of time.

[0183] At any time between introduction 107 of the specimen 104 and incubation 115, the specimen can be mixed with a volume of growth medium within the test device 109. The incubation 115 step can last less than or longer than about 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, or 24 hours, with about 4 hours being preferred to allow for measurable differential growth of target cells or microorganisms in the specimen 104. The leftmost track shows the initial number of target cells (n t0 ) is imaged to determine the amount of chromatin that has been incubated.

[0184] After incubation 115, the incubated portions 117 can be exposed to, for example, magnetic tags and detectable labels that can form complexes 119 with specific target cells in the specimen portion for imaging 127. The labeled target complexes 121 can then be magnetically deposited 123 on a detection surface. Imaging 127 of the deposited target complexes 125 in the portions can be performed and the images 129 can be analyzed by the instrument's image analysis software to quantify the amount of target 121 detected in each portion.

[0185] By comparing the amount of target cells or microorganisms in the portions incubated in the presence of various antimicrobial agents, it is possible to determine which of the agents inhibited growth. The comparison is made with the amount of target cells (n t0 ) is preferably included.

[0186] The systems and methods of the present invention can be used to identify appropriate therapeutic treatments for pathogens, including bacteria, fungi, parasites, and viruses. For these applications, target pathogens are incubated with various therapeutic agents under conditions that result in reduced pathogen viability in the absence of the therapeutic agents. Potentially effective agents for treating infections caused by target pathogens are identified by determining which agents negatively affect target viability, as evidenced by phenotypes indicative of reproduction, replication, growth, or these qualities and behaviors. Target bacterial pathogens can be incubated in the presence of various antimicrobial agents, for example, in nutrient bacterial growth media, and the effects of the agents on target bacterial growth in specimens can be analyzed to determine the effectiveness of antimicrobial agents.

[0187] FIG. 7 is a perspective view of the analytical cartridge 201. The specimen can be inserted directly into the specimen well 203. To distribute the specimen to the wells in the analytical cartridge 201, the air port 217 can be opened to apply pressure or vacuum. The specimen can be first distributed from the specimen well 203 to the partition well 203, held there by the valve 207 during incubation, released into the reagent well 209 for labeling, and then released into the imaging well 211 for imaging. The fluidic module of the instrument can be interfaced with the cartridge to control the movement of the valve in coordination with the specimen and reagent distribution according to the requirements of a particular test. The analytical cartridge 201 can have one or more identifiers 219 (e.g., bar codes) that, when analyzed or read by the instrument or a reader in the instrument, can include information about the patient and specimen to associate the cartridge with a set of instructions for processing in the instrument and to associate the test results with a particular patient.

[0188] The differential growth of microbial pathogens after incubation in the split wells 205 containing growth medium and antimicrobial agents is useful for determining antimicrobial susceptibility testing. In various embodiments, a portion of the specimen can be incubated in the split wells 205 for less than about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, or 24 hours before processing and imaging in the imaging wells. The growth medium and / or antimicrobial agent can be selected based on the target cells or microorganisms to be analyzed in the specimen and included in the test device 201. For example, a growth medium known to support the growth of the identified target cells or microorganisms, and a therapeutic agent or antimicrobial agent commonly used to treat the identified target can be selected. In various embodiments, the test device 201 can be pre-populated with growth medium and therapeutic agent for a particular target, so that a user who has identified a target in a specimen (e.g., determining the cause of a patient's infection) can select the appropriate pre-packaged test device for antimicrobial susceptibility testing of the target microorganism or therapeutic efficacy analysis of the target cells.

[0189] After a suitable incubation period, the valve 207 can be manipulated to move a portion of the incubated specimen from the partition well 205 to the reagent well 209. The treated 209 well can be pre-filled with treatment reagents to label target cells or microorganisms for imaging. The treatment reagents can be lyophilized or air-dried for storage and can be activated upon contact with a portion of the fluid specimen and growth medium. The reagent well 209 can contain, for example, a target-specific detectable label and a magnetic tag. As the portion of the incubated specimen passes through the reagent well 209, a target-specific complex is formed that is comprised of the target cell, the detectable label, and the magnetic tag.

[0190] The test device 201 is then subjected to a magnetic field (e.g., placed on a permanent magnet) to attract the complexes onto the detection surface at the bottom of the imaging well 211. The imaging well 211 may contain a dye cushion that forms a dense opaque water layer that underlies the upper assay layer in the imaging well described herein. All magnetic particles, including the target complexes, which are magnetically tagged fluorescently labeled target complexes, are attracted by the magnetic field through the lower dye cushion layer and deposited on the detection surface 215 of the imaging well 211, thereby carrying the labeled target cells or microorganisms through the dye cushion layer to the detection surface 215 of the imaging well 211. The detection surface 215 may be optically transparent to allow optical detection of the labeled target cells, molecules, viruses, or microorganisms. After processing and magnetic selection, the test device 201 can be placed on an imaging stage for image processing as described below.

[0191] The test device can have any number of divided wells and corresponding processing and imaging wells. Although the system and method of the present invention are described herein mainly in terms of a test device with interconnected wells for automated processing, the technique can also be implemented in any known fluid platform, including manual processing in the wells of a microtiter plate or on a substrate that also has droplet capabilities. In some embodiments, the number of wells is preferably large (e.g., 100 or more), limited only by the size constraints of the test device and the equipment.

[0192] By quantifying the amount of target cells, molecules, viruses, or microorganisms present in each of the specimen portions after incubation in the presence of various antimicrobial agents and comparing to an unincubated growth reference (nt0) obtained from the specimen prior to incubation, the effect of each therapeutic agent on target cell, molecule, virus, or microorganism growth can be determined. By determining which antimicrobial agent best inhibited growth, an effective therapy for treating the patient's infection can be determined.

[0193] The cartridge is used to process assays to detect specific molecular targets, specific cellular targets, and to determine effective treatments. When detecting specific microscopic or submicroscopic cellular or molecular targets, target-specific optical probes, such as fluorescent probes, are designed to bind to specific cellular or molecular targets. Target-specific magnetic particles are also designed to bind to cellular or molecular targets. A sample potentially containing the target is first mixed with the labeled probes and particles in the cartridge. The cartridge is incubated for a period of time to form complexes, each of which is composed of a target, an optical probe, and a magnetic particle. A magnetic field is applied to pull the magnetic particles and the remainder of the complexes down through a dye cushion. The optical probes bound to the target fluoresce, and imaging is used to identify the presence of the target cell or molecule. The imaging technique detects targets labeled with the optical probes without the use of magnification. Illuminating the labeled targets causes them to fluoresce. The emitted photons strike the CMOS chip of a digital camera, which contains an array of independent light-sensitive pixel elements. The pixel elements directly overlying each target are "illuminated" as white spots in the resulting image. A computer instantly enumerates the illuminated pixels, indicating the number of targets present. At low analyte concentrations, digitally counting individual labeled targets produces a better signal-to-noise ratio compared to the more common method of integrating the signal over a detection area. Non-magnified imaging allows for instantaneous imaging of a large field of view - allowing for the rapid detection of small numbers of cellular targets in a large volume specimen.

[0194] FIG. 8 is an example of a workflow for performing antimicrobial susceptibility testing of a specimen containing bacteria. The workflow illustrates incubation and processing steps that can be performed in an analytical cartridge according to various methods of the invention. The specimen can be mixed with a growth medium 501, which can be selected based on knowledge of the target microorganism present in the specimen (e.g., cation-adjusted Mueller-Hinton Broth selected to analyze E. coli in the specimen). The specimen and growth medium can then be divided 503 into a number (n) of split wells for incubation 507. One or more of the split wells can contain different antimicrobial agents. One or more portions can be analyzed without incubation 505 to quantify the target microorganism, for comparison to a portion of the specimen analyzed after subsequent incubation in the presence or absence of an antimicrobial agent. Incubation in the absence of a therapeutic agent can provide important information, including indications of non-viable microorganisms that may arise if the patient has already been treated. Analysis of such incubated portions can also serve as an internal control for growth inhibitory interference and reagent stability.

[0195] It is preferred that the target cells, molecules, viruses, or microorganisms have already been identified, and therefore various antimicrobial agents have been selected, for example, as antimicrobial agents commonly used to treat the identified targets. Each split well may contain a single antimicrobial agent, or a combination of antimicrobial agents to evaluate the effect of the combination on the target cell, molecule, virus, or microorganism growth. Different concentrations of different antimicrobial agents may be combined with portions or aliquots of specimens in different split wells, and some split wells may be used to replicate treatments with the same antimicrobial agent to enhance results. Some portions or aliquots of specimens in some split wells may not be combined with antimicrobial agents to quantify growth without inhibition and / or detect growth inhibition due to either non-antimicrobial inference or reagent instability.

[0196] After incubation 507, the incubated specimen aliquots can be processed and imaged in a similar manner as the t0 specimen aliquot 505, and the bacterial or other microbial counts can be compared to each other, to the t0 specimen aliquot, or to other control counts 509 to determine the effect of antimicrobial agents or combinations thereof. For example, the antimicrobial agent in Abx well 2 inhibited growth while other antimicrobial agents did not. Thus, the antimicrobial agent from Abx well 2 is recommended for treatment of the patient from whom the specimen was obtained.

[0197] A variety of instruments or devices can be used to interact with the test device to perform target identification and antimicrobial susceptibility testing of specimens, among other tests. The instrument may include an input mechanism for receiving and cataloging the test device. The test device may include one or more identifier tags (e.g., bar codes) readable by the instrument to determine application-specific test device type and specimen and patient information.

[0198] The instrument is preferably controlled by a computer to automate the manipulation of the test device, the identification of target cells, molecules, viruses, or microorganisms, and the performance of antimicrobial susceptibility testing analysis, as well as to generate and analyze imaging results. The instrument can be coupled to a user interface, such as a touch screen, to display prompts and results and receive commands. The instrument may include a conveyor or robotic arm for moving the test device within the instrument. To perform the steps required for target identification, the instrument preferably includes a magnetic module having a permanent magnet or electromagnet, for example, to provide a magnetic field for depositing the complex of the magnetic particle and the labeled target on the detection surface to be imaged. The instrument may also include an imaging module for capturing images of the labeled target cells, such as those described in U.S. Pat. Nos. 9,643,180 and 8,021,848, the contents of each of which are incorporated herein by reference, as well as a stage for manipulating the detection surface of the test device relative to the imaging module of the instrument. The imaging module can be operatively associated with a computer to provide image processing, analysis, and display capabilities. The instrument may also include a disposal module for disposing of the test device after use.

[0199] For antimicrobial susceptibility testing analysis, the instrument may include one or more incubation areas for storing the test devices during incubation for growth and / or test incubation. The incubation area may include heating and / or cooling elements and a thermostat to control its elements so that the incubation area is maintained at a desired temperature for growing target cells or microorganisms (e.g., 35° C.) or a desired temperature for performing test incubation. The instrument may include a fluidic module for driving the movement of specimens and reagents within the test device by, for example, hydraulic, pneumatic or vacuum forces, or manipulation of valves, plungers, and actuators using functionality provided by mechanical means incorporated in the fluidic module. In some embodiments, the mechanical conveyor arm may be operable to manipulate the test devices between various functional modules, including incubation modules, fluidic modules, magnetic modules, imaging modules, and waste modules.

[0200] FIG. 9 shows an exemplary instrument for performing target cell, molecule, virus, or microbial identification and antimicrobial susceptibility testing on specimens in a testing device. The instrument includes a carousel, a mechanical conveyor arm, and an incubation module, a fluidic module, a magnetic module, an imaging module, and a waste module. The instrument 601 can be used to interact with the analytical cartridge to perform target identification, antimicrobial susceptibility testing of the specimen, or other testing. The instrument 601 includes at least one user interface 603 (e.g., a touch screen) for displaying prompts, results, reports, and receiving commands. The instrument 601 can be divided into different wells. The wells can include a carousel 605 for transport and incubation of analytical cartridges, an upper well 607 for housing processing and incubation instruments, and a lower well 609 for housing electronic, imaging, and pneumatic instruments. The disclosed devices and methods can be used to identify and quantify a variety of target cells, molecules, or microorganisms, including viruses, bacteria, fungi, parasites, human cells, animal cells, plant cells, to detect infection. Antimicrobial susceptibility testing can be performed on a variety of target cells, molecules, viruses, or microorganisms by adjusting the growth medium and antimicrobial agent for the target.

[0201] Detectable labels can include any suitable type of optically detectable label. Non-limiting examples of detectable labels include resonant light scattering particles and quantum dots. Detectable labels can include target-specific moieties that preferentially bind to target cells, molecules, microorganisms, viruses, host cells, or other non-bacterial molecules. Target-specific binding molecules can include, for example, antibodies that bind to target-specific antigens or nucleic acid (or nucleic acid analog) probes that are complementary to target-specific nucleic acid sequences.

[0202] The fluorophores on the separate fluorescent probes can have separate photon signals, and thus the fluorescent signals for different categories of target cells or microorganisms in the test can be differentiated by the separate photon signals. Imaging methods for distinguishing 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 in a single processing well and imaging well for the presence of multiple specific target cells, molecules, viruses, or microorganisms.

[0203] In certain embodiments, the detectable label and magnetic tag, and the target-specific binding molecule can be separate or combined / linked. In certain embodiments, the magnetic particle and the detectable label can be non-specific, such as, for example, avidin-coated magnetic particles and SYBR-green dye. For example, biotin-labeled target-specific antibodies (i.e., target-specific binding molecules) can be used to target specific cells or microorganisms, which can then be tagged with avidin magnetic particles for specific magnetic selection. All cells are labeled with SYBR-green, but only magnetically separated (i.e., biotin-tagged) targets are deposited on the detection surface for imaging.

[0204] In a non-limiting embodiment, the detectable labeling of microorganisms includes fluorescent in situ hybridization (FISH). In FISH analysis, a fluorescent probe comprising a nucleic acid (or nucleic acid analogue) probe portion and a fluorophore portion is used to bind to a target-specific nucleic acid sequence by reassociation, and thus the target can then be optically detected. For example, a probe targeting a target-specific 16S rRNA can be used to selectively label and detect a 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 herein by reference. For identification testing, several separate target-specific fluorescent probes can be used to independently identify multiple separate categories of targets in a single specimen. The separate fluorescent probes can include separate nucleic acid probe portions designed such that under test reassociation conditions, the nucleic acid probe portion of the fluorescent probe preferentially reassociates with target-specific cellular nucleic acid sequences for separate categories of target cells or microorganisms. A detailed discussion of probe reassociation can be found in US Patent Publication No. 2003 / 0228599, which is incorporated herein by reference.

[0205] In certain embodiments, FISH analysis for identification and / or quantification of target cells or microorganisms in a sample can be performed isothermally, without reagent exchange, and without cell fixation, allowing automated on-device processing from reaction initiation to imaging results in about 30 minutes or less.

[0206] Magnetic particles and application of a magnetic field can be used to physically separate bound and unbound detectable labels in solution without a washing step. As described in U.S. Pat. No. 9,643,180, the contents of which are incorporated herein by reference, a dye cushion layer can be used to optically isolate the sample matrix and unbound optical labels from the detection surface of the imaging well, thereby minimizing or eliminating imaging background. The dyes in the dye cushion layer are preferably selected to absorb the excitation and emission light used by the instrument for imaging. Thus, signals from unbound label moieties in the assay layer do not significantly interfere with detection of signals from labeled target cell, molecule, virus, or microbial complexes magnetically deposited on the detection surface. Similarly, the use of the dye cushion prevents any autofluorescence from the sample matrix, also contained in the assay layer, from significantly interfering with detection of signals from the deposited labeled target cell complexes. These attributes of the dye cushion may allow target cells or microorganisms to be detected without sample preparation by the user and without washing steps to remove unbound labels from the test device.

[0207] Digital imaging of labeled target cells, molecules, viruses, or microorganisms can be achieved using a digital imager. In the preferred case of fluorescent labeling, various lenses, illumination sources, excitation sources, and filters can be used. The imaging module can include any device that can generate digital images of detectably labeled target cells, molecules, viruses, or microorganisms in solution or attracted to a detection surface in a well or test device. The imaging module can 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.

[0208] Imaging can be performed under a single set of conditions or light sources, and filters and / or lenses can be changed between images to detect different optically distinguishable labels (e.g., different fluorescent probes corresponding to different target cells or microorganisms). The imaging techniques and instruments described in U.S. Patent Nos. 9,643,180 and 8,021,848, the contents of each of which are incorporated herein by reference, may allow for the observation and enumeration of individual cellular, molecular, or viral targets.

[0209] FIG. 10A is an exemplary top view of the functional layout 601 of the instrument. The instrument 601 may include an input mechanism 703 (e.g., a loading rack or tray) for receiving and cataloging multiple analytical cartridges. The instrument 601 may also include a carousel 605 and a mechanical conveyor arm 707 for receiving, moving and manipulating analytical cartridges within the instrument 705. The instrument 601 may also include a task scheduler. The instrument 601 is preferably computer controlled to automate the manipulation of analytical cartridges, the performance of microbial identification and antimicrobial susceptibility testing analysis, and the generation of results. The instrument 601 may include multiple subsystems for carrying out the methods of the present invention. FIG. 10B is a perspective view of the carousel.

[0210] The subsystems of the instrument 601 may include a pneumatic subsystem 709, a magnetic subsystem 711, an imaging subsystem 713, and a waste bin subsystem 715. The magnetic subsystem 711 may include, for example, a permanent magnet or electromagnet that provides a magnetic field for depositing the magnetic particle-target complexes on a detection surface of the analytical cartridge for imaging. The imaging subsystem 713 may be such as those described above in U.S. Pat. Nos. 9,643,180 and 8,021,848, the contents of each of which are incorporated herein by reference, and a stage for manipulating the detection surface 109 of the analytical cartridge relative to the imaging module of the instrument 601. The imaging subsystem 713 may be operatively associated with a computer to provide image processing, analysis, and display capabilities. The pneumatic subsystem 709 may be operable to drive movement of the analytes 104 and reagents within the analytical cartridge 109, for example, through 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 be incorporated into the pneumatic subsystem 709 to achieve movement of the analyte 104. The waste subsystem 715 may include a receptacle (e.g., a removable bin) for disposing of the analysis cartridge 109 after use.

[0211] The instrument 601 may also include one or more incubation areas for holding (or storing) analytical cartridges during incubation for growth and / or test incubation. The incubation areas may include heating and / or cooling elements and thermostats to control the elements thereof such that the incubation area is maintained at a desired temperature for growing target cells or microorganisms (e.g., 35° C.) or a desired temperature for performing test incubations. In various embodiments, tests performed on the instrument may be designed to be isothermal, thus requiring a single temperature, at which the interior of the instrument may be maintained. In such cases, simple storage slots adjacent to the carousel or the carousel itself may serve as incubation storage for the various incubation steps of the tests being performed.

[0212] In some embodiments, the mechanical conveyor arm mechanism 707 may be operable to manipulate the analytical cartridge 109 between various subsystems within the instrument 601. In some embodiments of the invention, the mechanical conveyor arm 707 moves each of the analytical cartridges 109 between the carousel 605 and the various subsystems of the instrument. The mechanical conveyor arm 707 applies a pulling or pushing force to move the analytical cartridge 109 to or from the carousel 605. The carousel 605 rotates to position the analytical cartridge 109 next to another one of the subsystems, and then the mechanical conveyor arm 707 applies a force to slide the analytical cartridge 109 onto the subsystem. The analytical cartridge 109 is not grabbed by the mechanical conveyor arm 707 or any other element of the instrument 601. Sliding or pushing the analytical cartridge 109 within the instrument 601 reduces exposure to debris. The various stations or subsystems within the instrument, as well as the carousel 605, include slots 717, 719 sized to receive and guide the cartridges 109 as they slide between the carousel 605 and the various stations. Rotation of the carousel 605 aligns the slots 717 on the carousel 605 with corresponding slots 719 on the stations, thereby forming a guide or track along which the cartridges 109 can be slid by the mechanical conveyor arm 707.

[0213] In some embodiments, the instrument includes a task scheduler for managing the analytical cartridges 109 within the instrument 601. The task scheduler is operable to control the movement, such as transportation and movement, of each of the analytical cartridges 109 between multiple subsystems. In some embodiments, the time each analytical cartridge 109 spends within a subsystem may also be managed by the task scheduler. The task scheduler may reserve time in the various subsystems as needed for each analysis of the analytical cartridge 109. In some embodiments of the invention, the task scheduler may manage the movement of the analytical cartridges 109 (i.e., the steps / parameters of the analysis to be performed) by identifying the contents of the cartridge.

[0214] The scheduler may include software stored in tangible non-transitory memory 1305 and operated by a processor 1303, as shown in FIG. 11. The processor may communicate with the instrument 601 and various motors and subsystems or stations thereon, for example, to operate the carousel and mechanical conveyor arm, and to control the imaging devices in memory 1305 and record images received from the imaging subsystems or stations. The processor 1303 and memory 1305 may comprise a computer 1301, which may also include input / output devices 1307, such as a monitor, keyboard, mouse, or touch screen. Such a computer 1301 may be connected to a network 1309 to allow for the receipt and transfer of data, including test results, to other connected devices.

[0215] In some embodiments, the instrument 601 may also include a reader operable to analyze one or more identifiers (e.g., barcodes) 219 located on the analytical cartridge 109. The content amount and required processing of the analytical cartridge 109 may be associated with the identifier 219 on the analytical cartridge 109. Each of the analytical cartridges 109 may include an identifier 109 readable by the instrument 601. The instrument 601 may read the identifier 219 via the reader and associate the identifier 219 with a specific set of instructions to execute for the task scheduler. The reader may be located outside the instrument 601 and may move the analytical cartridge 109 from the loading rack 703 to the carousel 605 after reading the identifier 219. The identifier 219 may be a barcode and may be specific / unique to the specimen in the analytical cartridge 109. The one or more identifiers may identify specimen information, patient information, test information, or a combination thereof.

[0216] Upon reading the identifier, the computer processor can access the test associated with that identifier (e.g., antimicrobial susceptibility test for E. coli). The processor can then determine a schedule for performing the necessary steps of the test, and upon initiation, can determine when each station or subsystem is required and how long it will take to complete the test. From its memory, the processor can access the schedules of other cartridges currently running in the instrument and compare the availability of various stations at the required time. Certain steps (e.g., incubation) may be flexible and the schedule may provide for different lengths that can be changed to accommodate other scheduled operations on other cartridges. If initiating a test at a certain time would result in an irreconcilable conflict for any of the subsystems or stations, the instrument may reject the cartridge and notify the user of an acceptable later time to initiate and run the test without conflict. In certain embodiments, the instrument may include two or more of either the subsystems or stations to avoid such conflicts. For example, a highly accessed station such as a fluidics module may warrant the inclusion of two or more subsystems or stations depending on the desired capabilities of the instrument.

[0217] 12A and 12B show an exemplary mechanical conveyor arm 1901 according to one particular embodiment. The mechanical conveyor arm 1901 includes a rotatable shaft 1905 that is slightly longer than the length of the cartridge 109 to be manipulated. The mechanical conveyor arm 1901 also includes one prong 1903 positioned at each end of the rotatable shaft 1905. The mechanical conveyor arm 1901 can rotate the prong 1903 between a raised position shown in FIG. 12A and a lowered position shown in FIG. 12B. While in the raised position, the mechanical conveyor arm 1901 can be positioned above the cartridge 109 to be manipulated, and once in the proper position, the prongs 1903 can be lowered as seen in FIG. 12B to flank either side of the cartridge 109 to be manipulated. The movement of the mechanical conveyor arm 1901 then causes one of the prongs 1903 to contact one side of the cartridge 109 and the force of the movement of the mechanical conveyor arm 1903 is transferred to the cartridge 109. Due to the length of the rotatable shaft 1905 and the separation of the prongs 1903, only one prong 1903 can contact the cartridge 109 at a time, therefore the cartridge is not gripped or compressed by the instrument. As described above, the carousel and station slots act as tracks to guide the lateral movement of the cartridge 109, therefore only a single point of contact with the mechanical conveyor arm 1901 is required for the movement of the cartridge 109.

[0218] FIG. 13 shows an exemplary instrument 601 for use in the methods of the disclosure. The instrument 601 includes a user interface 603 for receiving user input and displaying results, status, and other information. To maintain a desired incubation or reaction temperature within the instrument 601, an enclosure is provided. The instrument has an access donor that opens to allow access to a loading tray 703 where a user can load a cartridge for analysis. The instrument 603 can read the identifier on the cartridge in the loading tray 703 before opening the inner door to begin processing and placing the cartridge into the carousel. That way, any errors or scheduling conflicts, if any, can be addressed before the cartridge is installed. The loading tray 703 positions the cartridge in a set position relative to the instrument and allows the instrument to scan a known position for the identifier and mechanical conveyor arm to mate with the cartridge and place it into the carousel. To avoid errors such as jamming or the identifier pointing away from the scanner of the instrument, the cartridge and loading tray may include an asymmetric footprint, so that the cartridge can only be inserted into the tray one way.

[0219] Figure 14 shows a cartridge 109 loaded into a loading tray as well as a cartridge 201 loaded with a specimen 104 (e.g., a urine specimen) obtained from a patient. After the specimen is loaded into the inlet 203 of the cartridge 201, the cartridge 201 is loaded into the loading tray and inserted into the instrument. The specimen loading step shown in Figure 14 is the only user interaction required, as all reagents for the test are preloaded into the cartridge and the instrument is operable to automatically perform all testing steps and provide actionable results. Thus, the opportunity for user error is reduced over standard laboratory techniques that require frequent user intervention.

[0220] 15 shows a user loading cartridges 109 into a loading tray 703 that has been placed in a receiving area of ​​the instrument 601. The interior door of the instrument 601 is closed and can remain closed until the instrument 601 recognizes the loaded cartridges 109 in the loading tray 703, scans them, and is ready to process them.

[0221] 16 shows a view of the interior of an instrument 601, which includes a carousel 605. The interior door of the instrument 601 is open and cartridges are slid by a mechanical conveyor arm onto the carousel for processing.

[0222] FIG 17 shows a side view of the internal components of the instrument according to an embodiment of the invention. A waste bin 2205 is located below the carousel assembly 605, an incubation chamber 2210 is located distal to the carousel assembly 605 in the upper portion of the instrument, and an electronics section 2215 is located distal to the carousel assembly 605 in the lower portion of the instrument. FIG 18 and FIG 20 show side views of the instrument according to an embodiment of the invention. FIG 19 shows a front view of the instrument according to an embodiment of the invention, showing the display panel or user interface 603. FIG 21 shows a rear view of the instrument according to an embodiment of the invention.

[0223] 22 shows one embodiment of a top view of the instrument layout. Cartridges are loaded into the instrument at a cartridge loading rack 703. A carousel assembly 605 is located distal to the cartridge loading rack 703 and centrally within the instrument. A fluidics module 2220, a magnetic module 2230, and an imaging module 2240 are located distal to the carousel assembly 605. A mechanical conveyor arm 2250 transports the cartridges within the instrument.

[0224] FIG. 23 shows an embodiment of the carousel 605. The carousel 605 is disposed on the instrument deck 2260. The mechanical conveyor arm 2250 transports the cartridges within the instrument and includes an R-theta transport mechanism 2253 and an arm extension drive 2257. FIG. 24 shows an embodiment of the R-theta transport mechanism 2253 of the mechanical conveyor arm 2250. The R-theta transport mechanism 2253 has cartridge transport fingers 2259 and transports the cartridges within the carousel 605. FIG. 25 shows an embodiment of the arm extension drive 2257 of the mechanical conveyor arm 2250. The arm extension drive 2257 transports the cartridges from the carousel 605 to the fluidics module 2220, the magnetic module 2230, and the imaging module 2240.

[0225] FIG. 26 illustrates an embodiment of the imaging module 2240. The imaging module 2240 includes an XYZ stage assembly 2270, which moves the cartridge in the x, y, and z directions for positioning during imaging, and is disposed above the instrument deck 2260. An optical assembly 2280 is disposed below the instrument deck 2260. FIG. 27 illustrates a top view of the imaging module 2240, including the instrument deck 2260, the XYZ stage assembly 2270, and a cartridge nest 2275. A cartridge is placed in the cartridge nest 2275, which is then moved by the XYZ stage assembly 2285 for positioning above the optical assembly 2280 for imaging. FIG. 28 illustrates an embodiment of the optical assembly 2280, which includes a light 2282, a lens 2284, a filter wheel 2286, and a camera 2288.

[0226] Figure 29 shows a top view of an embodiment of a fluidic module 2220. A cartridge is inserted into the cartridge slot 2224. A heater block 2223 is located opposite the cartridge slot 2224. A cartridge clamping mechanism 2221 having a cartridge clamp 2222 is used to clamp the cartridge into the cartridge slot 2224 and into contact with the cartridge valve actuator 2225. Figure 30 shows a side view of an embodiment of a fluidic module 2220 including cartridge valve push fingers 2227 and cartridge air port 2228.

[0227] Figure 31 shows a top view of an embodiment of a magnetic module. Cartridges slide into cartridge loading slots 3100. Each cartridge loading slot has a magnet 3120 disposed on the bottom surface of the cartridge loading slot, and each slot has a magnet shroud 3130 disposed around the perimeter of the magnetic slot. Figure 32 shows a side view of an embodiment of a magnetic module.

[0228] The systems and methods of the invention may include a computer operable to control the instrument and test device and / or to process the imaging results. 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 test device in the instrument and to obtain and process images of the labeled target cells.

[0229] 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).

[0230] Memory refers to a device or a system of devices that stores data or instructions in a machine-readable format. Memory may include one or more sets of instructions (e.g., software) that can achieve some or all of the methods or functions described herein when executed by one or more of the processors of the disclosed computer. Preferably, the computer includes non-transitory memory, such as a solid-state drive, a flash drive, a disk drive, a hard drive, a subscriber identity module (SIM) card, a secure digital card (SD card), a micro SD card, or a solid-state drive (SSD), optical and magnetic media, etc., or a combination thereof.

[0231] An input / output device is a mechanism or system for moving data into or out of a computer. Exemplary input / output devices include video display devices (e.g., liquid crystal display (LCD) or cathode ray tube (CRT)), alphanumeric input devices (e.g., keyboards), cursor control devices (e.g., mice), disk drive devices, signal generating devices (e.g., speakers), touch screens, accelerometers, microphones, cellular radio frequency antennas, and network interface devices, which may be, for example, network interface cards (NICs), Wi-Fi cards, or cellular modems. Input / output devices can be used to allow a user to control the instrument and receive data obtained by the instrument from the test device. EXAMPLES

[0232] (Example 0) overview Microtiter plates for pathogen identification and antimicrobial susceptibility testing The system and method of the present invention allows for pathogen identification (ID) and antimicrobial susceptibility testing (AST) across the full spectrum of infectious diseases, including healthcare associated infections (HAIs), such as sepsis, catheter associated urinary tract infections (CAUTI), ventilator associated pneumonia, and surgical site infections. The present invention achieves rapid results by enumerating low levels of pathogens directly from patient specimens, avoiding the days required for current culture methods. The present invention achieves high sensitivity and quantification by using digital non-magnified imaging to identify and count pathogen cells labeled with specific fluorescent nucleic acid probes. Once identified, the present invention uses robust phenotypic AST methods to determine appropriate targeted treatment. The test detects infections in about 30 minutes, identifies pathogens, and delivers susceptibility profiles in about 4 hours. The testing workflow is similar to automated identification and antimicrobial susceptibility testing, except that patient specimens are tested directly and the time-consuming culture-based colony purification steps are eliminated. The present invention eliminates cell lysis, amplification, biochemical purification, and wash steps. The method requires little or no sample preparation. Other advantages include the ability to deliver test results for polymicrobial infections, high throughput (>80 tests / shift), and a fraction of the cost of competing rapid methods. Thus, there is a need for diagnostic tests that can rapidly detect infections, identify pathogens, and determine appropriate targeted antimicrobial treatments.

[0233] ID / AST platform for rapid detection of symptomatic infections and determining targeted treatment. The ID / AST platform addresses gaps in current culture-based technologies by providing a test that rapidly and cost-effectively identifies patients with severe symptomatic infections, identifies pathogens, and determines appropriate targeted antimicrobial treatment. The ID test of the present invention is quantitative in nature, and the AST test of the present invention uses a robust phenotypic method that evaluates growth in a series of antimicrobial dilutions. However, in contrast to culture, the method of the present invention can detect infections, identify pathogens, and deliver AST results in about 30 minutes and 4 hours, respectively. The speed of the test is due to (1) the ability to test specimens directly, avoiding time-consuming colony purification steps; (2) the ability of the technology to rapidly enumerate small numbers of pathogen cells using non-magnifying digital imaging technology; and (3) the ability to assess pathogen susceptibility to antimicrobial drugs with very little bacterial production.

[0234] Platform Overview: The ID / AST platform provides rapid ID / AST results so that patients with severe symptomatic infections receive reasonable targeted treatment at the onset of infection. The automated ID / AST instrument accommodates a menu of application-specific microtiter plate-based ID and AST consumables.

[0235] To detect infection and identify pathogens, patient specimens are analyzed in the ID consumables to first determine if infection is present and, if so, identify the pathogen using digital imaging of fluorescently labeled cells. Once infection is detected and the pathogen identified, another aliquot of the same specimen is incubated in the AST consumables in nutrient medium containing various dilutions of the relevant antimicrobial. A susceptibility profile is established by determining the concentration of antimicrobial at which the pathogen grows.

[0236] Technical Advantages. Advantages of the present invention for symptomatic infectious disease ID / AST testing include: rapid infection detection and pathogen ID: approximately 30 minutes; rapid, robust, comprehensive phenotypic AST: approximately 4 hours; ID / AST results on non-sterile specimen types; ID and AST results on polymicrobial infections; low cost consumables and reagents; simple affordable equipment; no or minimal specimen preparation, robust to specimen matrix; ultrasensitive pathogen detection and enumeration; high throughput (>100 specimens / shift / instrument); fully automated specimen tracking (no typing required); and internal controls for accuracy.

[0237] In the present invention, individual microscopic or submicroscopic targets are detected without magnification. The imaging technique of the present invention detects target cells labeled with fluorescent oligonucleotide probes without the use of magnification. Illuminating the labeled cells causes them to fluoresce. The emitted photons strike a digital camera CMOS chip that contains an array of independent light-sensitive pixel elements. The pixel elements that lie directly above the individual targets are "illuminated" as white spots in the resulting image. A computer instantly enumerates the illuminated pixels, indicating the number of targets present. At low analyte concentrations, digitally counting the individual labeled cells results in a better signal-to-noise ratio compared to the more common method of integrating the signal over a detection area. Non-magnified imaging allows for real-time imaging of a large field of view - allowing for rapid detection of a small number of cellular targets in a large volume of specimen.

[0238] FIG. 33 shows how the present invention rapidly and specifically counts target cells in complex specimens without specimen preparation or washing steps. A liquid specimen potentially containing a target pathogen is first mixed with a fluorescent in situ hybridization (FISH) labeling reagent. The reagent includes a fluorescently labeled target-specific oligonucleotide probe that binds to ribosomal RNA (rRNA), a cell permeabilization reagent, and positively charged magnetic nanoparticles that bind to negatively charged bacterial cells. The mixture is added to a clear-bottom imaging well, the bottom surface of which is coated with a dry dye cushion reagent composed of a dye and a density agent. When the dye cushion specimen contains water, two layers are formed: an assay layer in which the target cells bind to the magnetic particles and are labeled by the fluorescent probe, and an underlying dense opaque dye cushion layer. By placing the imaging well over a permanent magnet, the magnetic particles and any cells that are bound to them are attracted through the dye cushion layer and deposited on the bottom surface where the fluorescently labeled target cells are instantly imaged and counted. The dye cushion approach optically isolates the analyte and unbound fluorescent reagent from the imaging surface, thereby eliminating assay wash steps and liquid handling by the instrument, thus significantly improving ease of use and reducing the cost and complexity of instrument operation.

[0239] Microtiter plate format allows for determination of minimum inhibitory concentrations (MICs) for multiple antimicrobials in multiple wells, as well as a consumable pipeline of testing for a broad range of symptomatic infections, pathogens, and antimicrobials. Specificity, efficiency of probe design, and cost-effectiveness are achieved using FISH-based ID. A simple, efficient, and cost-effective approach for magnetic selection uses cationic paramagnetic particles that bind to negatively charged bacterial cells. Simple and proven instrumentation streamlines product development and reduces costs.

[0240] CAUTI ID Test. The CAUTI ID test determines if a patient has a UTI caused by one or more of the common CAUTI pathogens. Table 1 lists the pathogens identified by the test. Together, these pathogens account for 95% of CAUTI cases in the United States. Specimens with pathogen counts greater than 10,000 CFU / ml are scored as positive.

[0241] Table 1: Pathogens identified [Table 1-1] [Table 1-2]

[0242] Figure 34 shows the workflow of CAUTI ID testing. Urine samples are first mixed with the above FISH reagents without target-specific oligonucleotide-labeled probes. The mixture is then added to the wells of a CAUTI ID imaging plate, with each well containing a fluorescent nucleic acid probe targeting a different CAUTI pathogen. After FISH labeling and magnetic selection (approximately 30 minutes), the wells are imaged on the ID / AST instrument.

[0243] To assess total bacterial bioburden, the CAUTI ID test also enumerates microbial cells using DNA staining. The test includes internal positive and negative controls that demonstrate assay efficacy using both Gram-positive and Gram-negative targets.

[0244] CAUTI AST Testing. The AST method evaluates the ability of pathogens identified by the CAUTI ID test to grow in the presence of antimicrobials. Figure 35 shows the workflow of CAUTI AST testing. The specimen is first mixed with growth medium and then split into multiple growth wells containing dilutions of the antimicrobial relevant to that pathogen. Two microtiter plate-based panels were developed: CAUTI AST for Gram-negative pathogens. GN Panel and CAUTI AST GP Panel for Gram-positive Pathogens. The panels contain dried serial two-fold dilutions of antimicrobial agents. Candidate antimicrobial agents for the panels are listed in Table 2.

[0245] Table 2: Candidate antimicrobial drugs [Table 2]

[0246] For each antimicrobial, the number of target cells in the various wells is enumerated after selective growth at about 35° C. to determine the minimum inhibitory concentration (MIC). To assess differential growth, pathogen-specific fluorescent probes and FISH reagents corresponding to the identified pathogens are combined with the contents of each well and transferred to an imaging plate containing a dry dye cushion. After incubation and magnetic selection, the ID / AST hospital instrument enumerates the number of target cells in each well and then determines the MIC for each antimicrobial.

[0247] Instrumentation: The platform is a semi-automated system that includes an automated ID / AST hospital instrument, a magnetic station for microtiter plates, and an off-the-shelf 96-well pipettor for liquid transfer. The ID / AST instrument and magnetic station are based on current automated imaging instruments and a magnetic station for analyzing microtiter plates.

[0248] Feasibility Data. Development of a rapid, simple, high-throughput FISH assay for pathogens. A 30-minute FISH-based assay for pathogens was developed. All steps proceed in a single assay mix and the method does not require any washing steps. Coordinated cell permeabilization, hybridization / labeling, and binding to magnetic particles are followed by magnetic selection and imaging. The method uses inexpensive and readily available reagents such as unmodified magnetic particles, oligonucleotides, and surfactants.

[0249] By optimizing the probe design, permeabilization and hybridization conditions, we developed a labeling method that significantly improved FISH signal intensity. The method uses multiple labeled target-specific oligonucleotide probes and a helper probe that assists in unwinding rRNA secondary structures. Detection of eight target CAUTI pathogens at the required threshold (10,000 CFU / ml) was demonstrated in just 30 minutes directly in urine samples (Escherichia coli, Enterococcus faecalis, Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus mirabilis, Staphylococcus aureus, Streptococcus agalactiae, and Enterobacter pneumoniae). We developed a method for enumerating total bacterial counts in urine specimens using DNA staining, which is valuable for ruling out infections and identifying polymicrobial specimens and asymptomatic UTIs.

[0250] Limit of detection of pathogens in urine. Figure 36 shows that the limit of detection (LoD) of the new method is approximately 170 cells / assay for E. coli spiked into an assay containing 10% urine. This is significantly below our target LoD of 250 cells / assay that would be required to meet a positivity threshold of 10,000 pathogen cells / mL in 10% urine (assuming an assay volume of 250 μL). Furthermore, having demonstrated assay robustness in assays containing up to 70% urine, we expect the LoD to be significantly improved by using higher concentrations of urine (e.g., 20% or more).

[0251] FISH assays must be comprehensive, i.e., detect all strains of a given target pathogen. Figure 37 shows representative comprehensiveness data for E. coli, demonstrating that the new method can achieve high comprehensiveness with respect to the target pathogen. The specificity of the method was also demonstrated. The pathogen-specific assay does not produce false positives due to cross-reactions with other pathogens or commensal microorganisms in the specimen. Representative specificity data in Figure 38 demonstrates the excellent specificity that can be achieved with the method.

[0252] The CAUTI ID results in Figure 39 demonstrate the potential for achieving accurate identification. A microtiter plate test format was used with different target specific probes in different wells (columns). Urine specimens spiked with different CAUTI pathogens (rows) were tested. The tests were processed and analyzed using the company's prototype for the proposed instrumentation.

[0253] Figure 40 shows CAUTI AST test results for E. coli and E. faecalis, the most common Gram-negative and top Gram-positive pathogens causing CAUTI. Results demonstrated essentially 100% agreement of MICs between the 4-hour CAUTI AST method and the traditional broth microdilution reference test. Other results not shown here demonstrated a dynamic range of over 4 orders of magnitude and at least 12 months of room temperature stability of the microtiter plate consumables with dry dye cushion. These data demonstrate the feasibility of developing a rapid, sensitive, multiplexed UTI test that delivers accurate phenotypic antimicrobial susceptibility results in hours directly from patient specimens.

[0254] Example 1 Limits of detection (LoD) for Gram-negative bacteria using a new rapid fluorescent in situ hybridization assay

[0255] 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:

[0256] 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.

[0257] 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 ...

[0258] 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 minutes 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 44.

[0259] 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).

[0260] 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".

[0261] 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. The instrument uses a high-precision linear stage from Prior Scientific (Rockland, MA) to position each well above 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 fluorescent 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-bit quantization per pixel. A final image for each well is then formed by summing multiple frames. Sixteen frames were captured with a 100-millisecond exposure using a 635 / 25-nm excitation and 680 / 40-nm emission filter. Imaging of focal particles is performed with 470 / 40 nm excitation and 520 / 40 nm excitation filters, and 2 frames are captured with a 20 ms exposure.

[0262] Data analysis: At each cell concentration, the number of fluorescent objects detected was determined. For each bacterium tested, data from all eight cell concentrations were fitted to a linear regression line and the upper limit of blank (LoB) and limit of detection (LoD) were determined using the slope, intercept and standard deviation of a minimum of three cell inputs. result:

[0263] Low limits of detection were demonstrated for all three bacteria tested. The figures show data generated for E. coli, K. pneumoniae, and P. aeruginosa, and linear fitting was used to calculate the LoB and LoD. The LoB and LoD are shown in CFU detectable in a single reaction well.

[0264] Conclusions: The novel rapid FISH method described in this example is shown to be a highly sensitive method with a detection limit of approximately 500 CFU per reaction or less using minimally treated urine matrix.

[0265] Variations. This example illustrates the performance of this novel FISH method and is not limited to the specific details contained herein. Thus, those skilled in the art will 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, component concentrations), urine concentrations, and urine processing procedures. This methodology can also be clearly extended to other biological specimens and other bacterial and non-bacterial pathogens.

[0266] Figure 41 shows that the limit of detection (LoD) for E. coli ATCC 19138 is shown. The upper limit of blank (LoB) was 89 CFU / assay and the LoD was 284 CFU / assay, which corresponds to a LoD of 9,467 CFU / ml of urine.

[0267] Figure 42 shows that the limit of detection (LoD) for P. aeruginosa ATCC 9721 is shown. The upper limit of blank (LoB) was 104 CFU / assay and the LoD was 506 CFU / assay, which corresponds to a LoD of 16,867 CFU / ml of urine.

[0268] Figure 43 shows that the limit of detection (LoD) for K. pneumoniae ATCC 700603 is shown. The upper limit of blank (LoB) was 109 CFU / assay and the LoD was 319 CFU / assay, which corresponds to a LoD of 10,633 CFU / ml of urine.

[0269] FIG. 44 is a table of the probe sequences used in this example.

[0270] Example 2 Inclusiveness: Detection and identification of different strains of bacterial species using the present rapid FISH method

[0271] Overview: This example demonstrates the use of the present invention to detect different strains of targeted bacterial species. Raw data is presented for 11 different E. coli strains, and data is summarized for K. pneumoniae, P. aeruginosa, P. mirabilis and Enterococcus spp. 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.

[0272] Preparation of bacterial cells: Bacterial cultures of different strains were obtained by inoculating Trypticase Soy Broth (TSB, Hardy Diagnostics Cat. U65) with 3–5 colonies from fresh Tryptic Soy Agar plates (TSA, BD Cat. 221185) and growing for 1.5–3 h at 35°C to achieve log-phase growth. Optical density at 600 nm was used to estimate cell concentration, and cells were diluted to approximately 600 and 3000 CFU per reaction in 1× cation-adjusted Mueller-Hinton Broth (MHBII, Teknova Cat. M5860). For more accurate calculation of percent cell detection, 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 growth for 16–24 h at 35°C.

[0273] Preparation of magnetic particles: Magnetic particles conjugated with polyaspartic acid (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. 12Fluorescent magnetic microspheres containing a green dye (Dragon Green Fluorescent Microspheres, BANGS Laboratories, catalog MEDG001) were added to the suspension at a final concentration of 3 × 10 6 The magnetic particles were added at 1000x10000 particles / mL. These particles allow the optics to focus at the correct plane. The magnetic particle mixture was sonicated for 1 minute immediately before use to minimize clumping. Separate magnetic particle suspensions were prepared for the zero and 4 hour assays as described below.

[0274] Labeling of bacterial cells: 100 μL of the labeling reaction was added to the 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 targeting 16S or 23S bacterial rRNA (Integrated DNA Technologies, IDT), helper probes (IDT) to facilitate efficient hybridization, and pooled human urine (Innovative The probe sequences are shown in the table in FIG.

[0275] 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 the mixture. The final reaction mixture was transferred to a microtiter plate containing 50 μL of (pre-dried) "dye cushion" (50 mM TRIS pH 7.5 (Teknova 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 guide the magnetic particles, the fraction containing the labeled cells, through the "dye cushion" and into the vicinity of the imaging surface at the bottom of the well.

[0276] Imaging of labeled cells: Labeled bacterial cells with 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. The instrument uses a high-precision linear stage from Prior Scientific (Rockland, MA) to position each well above 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-bit quantization per pixel. A final image for each well is then formed by summing multiple frames. Sixteen frames were captured with a 100-millisecond exposure using a 635 / 25-nm excitation and 680 / 40-nm emission filter. Imaging of focal particles is performed with 470 / 40 nm excitation and 520 / 40 nm excitation filters, and 2 frames are captured with a 20 ms exposure.

[0277] 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 above 3 standard deviations of the signal in the cell-free condition. result.

[0278] Figure 41 shows the assay signal for 11 E. coli strains. All 11 strains were detected above background "no cells" conditions at a cell input of approximately 600 CFU per assay.

[0279] Figure 42 shows the data expressed as the percentage of cells detected (total assay signal in cell input wells - background assay signal / total cell input * 100). There was some variation in detection efficiency from strain to strain, but this did not inhibit the ability of the assay to detect each of the 11 different E. coli strains.

[0280] The table in Figure 47 summarizes the inclusion results for E. coli, K. pneumoniae, P. aeruginosa, P. mirabilis and Enterococcus spp., 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.

[0281] Conclusions: The novel FISH method described in this example detected all tested strains of five different bacterial species that are among the main pathogens leading to clinical symptoms in patients with UTI.

[0282] Variations. This example illustrates the performance of this novel FISH method and is not limited to the specific details contained herein. Thus, those skilled in the art will 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, component concentrations), urine concentrations, and urine processing procedures. This methodology can also be clearly extended to other biological specimens and other bacterial and non-bacterial pathogens.

[0283] Figure 45 shows that the average signal (n=3) for 11 E. coli strains is plotted for input cell concentrations of approximately 600 CFU / assay (light grey bars) and 3000 CFU / assay (dark grey bars). The signal from a no-cell control (blank) is shown on the left side of the figure. Error bars represent 1 standard deviation.

[0284] Figure 46 shows that the percentage of input cells detected (determined by plate count) is shown for each of the 11 E. coli strains. Each bar represents the average of six determinations, three from each of two different input cell levels. Percentage cell detection was calculated as [(assay signal-background signal) / input cells]*100.

[0285] FIG. 47 is a table showing inclusion results for four additional bacterial species.

[0286] FIG. 48 is a table showing the probe sequences used in this example.

[0287] Example 3 Specific detection of target bacteria using rapid isothermal FISH

[0288] Summary: This example demonstrates that a novel isothermal FISH method specifically detects target bacteria while not detecting related non-target bacteria, even at very high concentrations. This example presents 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.

[0289] Bacterial cell preparation: Bacterial cultures of 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 to fresh TSB and grown for 1.5–2 h until the optical density at 600 nm reached 0.15–0.3. Each bacterium was then diluted at approximately 1 × 10 per mL in cation-adjusted Mueller-Hinton (MHBII, Teknova catalog M5860). 8 The cells were diluted.

[0290] Selection of bacterial targets to evaluate: Bacterial pathogens to test for specificity were selected for sequence similarity of their rRNA to 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 is most problematic. The table in Figure 49 shows the bacterial species and strains tested.

[0291] Preparation of FISH probes: A DNA probe set for E. coli was heated in a water bath at 80-85 °C for 10 min, then placed on ice to reduce aggregation. This DNA probe set is shown in the table in Figure 50. The set contains species-specific DNA oligonucleotides labeled with a fluorescent dye (Alexa647N, Thermo Fischer) and helper oligonucleotides that are designed to bind to adjacent or nearby specific probes and disrupt local secondary structures of ribosomal subunits, allowing the labeled specific probes to have higher hybridization efficiency to the target rRNA.

[0292] Preparation of magnetic particles: Magnetic particles conjugated with polyaspartic acid (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 a green dye (Dragon Green Fluorescent Microspheres, BANGS Laboratories, catalog MEDG001) were added to the suspension at a final concentration of 3 × 10 6 The magnetic particles were added at 10 ...

[0293] Labeling of bacterial cells: 100 μL of the labeling reaction was added to the 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 targeting 16S or 23S bacterial rRNA (Integrated DNA Technologies, IDT), helper probes to facilitate efficient hybridization (IDT) and pooled human urine (Innovative The probe sets tested were prepared by combining 30 μL of 10 ... 6A final concentration of cells was tested, which is approximately 3000 times the detection limit determined for E. coli. After incubation, the microtiter plate was placed on a magnetic field (Dexter magnetic technologies, catalog 54170260) for 4 minutes to guide the magnetic particles, the fraction containing the labeled cells, through a "dye cushion" and into the vicinity of the imaging surface at the bottom of the well.

[0294] 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.

[0295] Data analysis: For each bacterium, the number of fluorescent objects was determined (assay signal). Bacteria were considered cross-reactive if the signal was detected within 3 standard deviations of the signal in the blank (no bacteria added). result.

[0296] The images show that the rapid novel FISH method detects only E. coli and not 16 other different challenge bacteria, and each of the images shows that very high concentrations (1 × 10 per reaction) of E. coli were detected. 6 It is shown that eight clinically relevant challenge bacteria (no cells) are not detected under the same assay conditions that yield a high assay signal for the E. coli targeted bacteria. The two bars represent two different probe sets designed to be specific for E. coli (see table in Figure 50). The assay signal for each of the 16 challenge bacteria was less than the no cells control plus three standard deviations (125).

[0297] Conclusion: The novel rapid FISH method described in this example is designed to specifically detect E. coli but not 16 clinically relevant potentially cross-reactive bacteria, demonstrating the high specificity of the method for identifying target UTI pathogens, which is crucial for the clinical treatment of infections.

[0298] 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 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, component concentrations), urine concentrations and urine processing procedures. This methodology can also be clearly 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.

[0299] FIG. 49 is a table showing the challenge bacteria to test the specificity of E. coli detection.

[0300] FIG. 50 is a table showing the probe sequences used in this example.

[0301] FIG. 51 shows the specific detection of E. coli and the absence of detection of the eight challenge bacteria.

[0302] FIG. 52 shows the specific detection of E. coli and the absence of detection of eight additional challenge bacteria.

[0303] Example 4 A multiplexed FISH assay for simultaneous identification of four distinct microorganisms.

[0304] 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 is labeled with four separate fluorophores with distinct excitation / emission spectral characteristics - Through the use of - one for each bacterial species - specifically detected in the mixture.

[0305] Experimental Methods. Bacterial cell growth: Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC 13883, Pseudomonas aeruginosa ATCC 27853 and Klebsiella Bacterial cultures of Ca. oxytoca ATCC 8724 were cultured using Trypticase Soy Broth (TSB, Hardy Diagnostics catalog U65) was inoculated with 3–5 colonies from a fresh Tryptic Soy Agar plate (TSA, BD catalog 221185) and grown at 37°C for 1.5–3 h to achieve log-phase growth. Each culture was then diluted to approximately 1.0 × 10 8The solution was diluted to an optical density at 600 nm of 0.15 colony forming units (CFU) / mL.

[0306] 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:

[0307] Labeling of bacterial cells: A 100 μL labeling reaction was prepared by mixing 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 Cat. C3649), 0.72% w / v SB3-12 (Sigma Cat. D0431), 0.13 M guanidine thiocyanate (Sigma A magnetic particle preparation was prepared by combining 0.18% w / v cetrimide (Sigma Cat. G9277), 0.18% w / v cetrimide (Sigma Cat. M7365), species-specific DNA probes targeted to 16S or 23S bacterial rRNA (Integrated DNA Technologies, IDT), a helper probe to promote efficient hybridization (IDT) and 30 μL of pooled human urine (Innovative Research, Cat. IRHUURE500ML). 10 μL of 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. 54.

[0308] 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).

[0309] 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. result.

[0310] Figure 53 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 those objects 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.

[0311] 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:

[0312] 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.

[0313] Figure 53 is an image showing the same field of view acquired in four different color channels using a CCD imaging method and four different fluorophores, one for each bacterium. All four bacteria could be detected in a single well.

[0314] FIG. 54 is a table of the probe sequences used in this Example 4.

[0315] Example 5 Specific detection of polymorphonuclear neutrophils (PMNs) using antibody-coated magnetic particles and fluorescently labeled antibodies

[0316] Overview. The presence and number of polymorphonuclear neutrophils (PMN) can be diagnostically informative for the detection of infection. For example, a low number of neutrophils in a urine sample helps to rule out urinary tract infection. In this example, non-magnified digital imaging was used to enumerate PMN targets stained with fluorescently labeled antibodies. In this example, anti-CD15 and anti-CD16 antibodies, which are directed against specific molecules present on the surface of PMN, were used for capture and detection. In one embodiment, the anti-CD15 antibody used for PMN capture was conjugated to magnetic particles, and the anti-CD16 antibody was fluorescently labeled for detection using non-magnified digital imaging. Experimental method. Blood samples

[0317] Fresh blood samples from healthy donors were obtained from Research Blood Components (Boston, Mass.) and used as a source of PMNs. Preparation of magnetic particles:

[0318] Antibody-conjugated magnetic particles were generated by coupling magnetic particles (Ademtec, 292 nm) to mouse anti-CD15 antibody (Biolegend) using standard EDAC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) coupling chemistry. Detection antibody

[0319] Alexa Fluor® 488 anti-human CD16 antibody (Biolegend) was used as the detection antibody. PMN labeling and capture:

[0320] The assay was performed in a 96-well microtiter plate. Each well contained 38 μl of phosphate-buffered saline (PBS), 2 μl of fresh blood sample or 2 μl of PBS (no PMN control), 5 μl of Alexa-488-labeled anti-CD16 antibody (1 μg) and 5 μl of antibody-conjugated magnetic particles (2e10 / mL). The reaction was incubated for 15 minutes at room temperature. After incubation, 40 μl of the reaction mixture was carefully layered on top of 75 μl of a "dye cushion" (15% Optiprep with 5 mg / mL Chromotrope 2R) that had been pre-dispensed in a black, clear-bottom half-area microtiter plate (Greiner 675096, VWR part#82050-056). The microtiter plate was placed over a magnetic field for 4 minutes to guide the magnetic particles, the fraction containing the labeled cells, through the "dye cushion" and into the vicinity of the imaging surface at the bottom of the well. Imaging of PMNs:

[0321] After magnetic capture of the labeled PMN:magnetic particle complexes, the microtiter plate was placed on a stage above a CCD digital camera (IDS, model UI-2250SE-M) and illuminated with light from an LED passing through an optical filter (469 nm, 35 nm FWHM). The fluorescent signal passing through an emission filter (520-35 nm) was detected by the camera to create an image of the fluorescent complexes. Images were analyzed using FLimage software (First Light Biosciences) to enumerate individual cells. result.

[0322] FIG. 88 shows the results of the MultiPath assay, which specifically detects PMNs present in the blood sample, while the detectable fluorescent signal of the buffer without the blood sample is very low.

[0323] Conclusion: This wide-range, non-magnifying imaging system is capable of detecting and enumerating PMNs from blood samples that are labeled with fluorescently labeled antibodies against cell surface markers. The results demonstrate the potential of the systems and methods of the present invention for enumerating diagnostically informative human or host cells.

[0324] Variations. Other cell-specific antibodies can be used to detect different cells in various biological samples. For example, other cell surface marker antibodies can recognize different diagnostically informative cells. For example, quantification of squamous epithelial cells is important for assessing the quality of respiratory samples in the diagnosis of pneumonia. Multiple antibodies / cell surface markers can be used, and labeled cells can be differentiated using multiple excitation and emission wavelengths for fluorescence detection. The spectrum of fluorescence related to the object can be used to determine whether it is a signal of a particular cell type.

[0325] Example 6 Automated Rapid AST of E. coli in Clinical Urine Specimens in On-Device Cartridges

[0326] 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:

[0327] 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.

[0328] 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.

[0329] 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. 6The magnetic particles were added to the suspension at a final concentration of 10 ...

[0330] 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.

[0331] 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.

[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] 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 antibiotics). Samples were grown for 4 hours at 35° C. in a standard incubator.

[0334] 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.

[0335] Comparison Methods: Results for the MulitPath™ assay were compared to broth microdilution (BMD) performed according to CLSI method M07-Ed13E 2018.

[0336] 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.

[0337] Figures 55 to 58 show three examples from our larger dataset that demonstrate how this method can be used to obtain MICs for urine of three individuals that match the gold standard broth microdilution method.

[0338] Figure 55 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 broth microdilution correspond exactly to the MICs determined by the growth fold threshold.

[0339] Figure 56 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 broth microdilution correspond exactly to the MICs determined by the growth fold threshold.

[0340] Figure 57 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 broth microdilution correspond exactly to the MICs determined by growth fold threshold.

[0341] Figure 58 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.

[0342] 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.

[0343] 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.

[0344] Figure 55 shows BIUR0017 containing nitrofurantoin.

[0345] FIG. 56 shows BIUR047 containing cefazolin.

[0346] Figure 57 shows BIUR057 containing ciprofloxacin.

[0347] FIG. 58 shows BIUR052 containing trimethoprim / sulfamethoxazole.

[0348] FIG. 59 is a table of the probe sequences used in this Example 6.

[0349] Example 7 Rapid and accurate antimicrobial susceptibility testing for bacteria in urine samples

[0350] 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.

[0351] 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.

[0352] 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.

[0353] 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 ...

[0354] 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. 63. 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, 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 (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.

[0355] 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.

[0356] 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.

[0357] 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.

[0358] 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.

[0359] Comparison Methods: Results for the MulitPath™ assay were compared to broth microdilution (BMD) performed according to CLSI method M07-Ed13E 2018.

[0360] 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.

[0361] Figure 60 further shows that for bacteria tested against ceftazidime (CAZ), the presence of exclusive filamentous bacteria (as easily identifiable visually by comparing the left (normal bacteria) to the 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.

[0362] Figure 60 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.

[0363] The table in Figure 62 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.

[0364] 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.

[0365] 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.

[0366] FIG. 60 is a visual comparison of normal bacteria (left panel) against filamentous fungi (right panel).

[0367] FIG. 61 shows that the MIC obtained by the novel, rapid AST method described in this invention is called at 0.25 μg / mL.

[0368] Table A: Bacteria used in this example and their previously determined antibiotic resistance (indicated with an "X") [Table A-1] [Table A-2] [Table A-3]

[0369] FIG. 62 is a table of AST results for all bacteria and antibiotics tested in this example.

[0370] FIG. 63 is a table of the probe sequences used in this Example 7.

[0371] Example 8 Rapid and accurate automated AST results on clinical urine samples without the use of cell purification

[0372] 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.

[0373] 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.

[0374] 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.

[0375] 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

[0376] A few days prior to preparing the cartridges (see the figure 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 4-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. For the final concentration of each antibiotic in the cartridge, see Table 1. The medium and antibiotics were then dried in a convection oven at 40 °C for 16-20 h. Preparation of AST Cartridges - Hybridization Reagents

[0377] 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 diagram for location on cartridge). Preparation of AST Cartridges - Magnetic Particles

[0378] 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

[0379] 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

[0380] 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 Table 2. 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

[0381] 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.

[0382] 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

[0383] 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.

[0384] 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. result.

[0385] Figure 65 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 matched these susceptibility / resistance calls.

[0386] The figure 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.

[0387] 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.

[0388] 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.

[0389] Figure 64 shows the Multipath™ UTI-AST cartridge.

[0390] FIG. 65 is a table showing the concentrations of antibiotics tested.

[0391] FIG. 66 is a table of the oligonucleotides used in this Example 8.

[0392] Figure 67 shows the results for BIUR0067.

[0393] Figure 68 shows the results for BIUR0084.

[0394] Example 9 Rapid AST methods directly on urine specimens are robust to a range of pathogen concentrations

[0395] 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.

[0396] 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.

[0397] 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.

[0398] 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 ...

[0399] 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.

[0400] 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. The instrument uses a high-precision linear stage from Prior Scientific (Rockland, MA) to position each well above 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 fluorescent 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-bit quantization per pixel. A final image for each well is then formed by summing multiple frames. Sixteen frames were captured with a 100-millisecond exposure using a 635 / 25-nm excitation and 667 / 30-nm emission filter. Imaging of focal particles is performed with 470 / 40 nm excitation and 520 / 40 nm excitation filters, and 2 frames are captured with a 20 ms exposure.

[0401] 4 hour growth: At the same time as the zero hour cell quantification, 10 μL of each organism inoculum, 10 μL of pooled urine, and 70 μL of 1×MHBII were added to the appropriate wells of the antibiotic plate (already containing 10 μL of antibiotic). Samples were grown for 4 hours at 35° C. in a standard air incubator.

[0402] Bacterial cell labeling during AST at 4 hours of growth: After samples were incubated with and without antibiotics for 4 hours (T4), cells were labeled and quantified to determine how much, if any, growth had occurred. 10 μL (10%) of the incubated sample-antibiotic plate was transferred to a microtiter plate and combined with 100 μL hybridization buffer, FISH probe, helper probe, magnetic particles, and focusing particles in the same manner as described above for the assay at time zero.

[0403] Comparison Methods: Results using the novel AST method described herein were compared to broth microdilution (BMD) performed according to CLSI M07-Ed13E 2018.

[0404] Data analysis and threshold generation: Using the image captured by the CCD camera, the detected cells were estimated by an algorithm that looked at both the number of objects in the field of view and the intensity of the objects. The number of cells based on this detection algorithm was generated at zero time, as well as at 4 hours without antibiotic and at 4 hours with each antibiotic at all concentrations. For each sample inoculum / drug concentration, the growth fold was calculated as the signal in the well containing the antibiotic after growth (4 hours) relative to the signal in the urine sample before growth (zero time). Using the growth fold and the observation of growth in the corresponding well in CLSI-compliant broth microdilution, a logistic regression model was used to generate a threshold value to determine the growth fold cutoff above which the cells grew in the presence of antibiotic (and thus were resistant at that concentration) and below which the cells were in the process of death (and thus were sensitive at that concentration). The point at which the number of growth folds fell below the determined threshold is the MIC value generated by this assay. The results were assigned to categories of sensitive, intermediate, or resistant to each antibiotic correspondingly. All data were then compared to the CLSI standard BMD. Four hours of growth in the absence of antibiotics is the control condition to ensure that viable bacteria are present in the processed urine sample. result.

[0405] The figure below shows how this method matches the gold standard broth microdilution method while also showing how robust it is to various inoculum levels.

[0406] In Figure 69, the results obtained using the new AST method are compared to those of the standard BMD performed at a single concentration for all drugs tested. The third column compares the MICs obtained by the new AST method to those obtained by the gold standard BMD. All MIC calls were within 1x2 dilutions of the CLSI-compliant BMD (essential agreement). The fourth column compares the categorical antibiotic susceptibility results (S=susceptible, I=intermediate, R=resistant) based on MICs (categorical agreement). A subset of Klebsiella concentrations showed categorical calls that differed from the broth microdilution MICs, but all of these were simply classified as minor errors by the standard AST methodology.

[0407] Figure 70 shows the MICs obtained using the novel 4-hour method (filled circles) for all inoculum levels for E. coli BAA-2469 compared to the standard broth microdilution method (24-hour BMD, dashed line). All MICs determined using the novel method were within essential agreement (shaded areas).

[0408] Figure 71 shows the raw data. Conclusion.

[0409] The rapid 4-hour AST method presented here is robust to initial cell concentrations over a wide range of target cell concentrations. Robustness to various inoculum concentrations is important for rapid AST methods because the target cell concentration is not known when testing specimens directly from the specimen.

[0410] Variations. This example illustrates the performance of this novel AST method and is not limited to the specific details contained herein. Thus, those skilled in the art will 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, component concentrations), urine concentrations, and urine processing procedures. This methodology can also be obviously extended for other biological specimens, as well as other bacterial and non-bacterial pathogens for which specific probes can be designed, as well as other antimicrobial or chemical agents.

[0411] Figure 69 is a summary of the overall intrinsic and categorical agreement for all organisms, antibiotics and inoculum levels.

[0412] FIG. 70 shows the MIC results for various inoculum levels generated using the new method described herein compared to the conventional BMD method.

[0413] FIG. 71 is a summary of the MIC results generated for various inoculum levels.

[0414] FIG. 72 is a table of the probe sequences used in this Example 9.

[0415] Example 10 Rapid antimicrobial susceptibility testing of target pathogens in clinical urine specimens containing multiple bacterial species without cell purification

[0416] Overview: Current methods of 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, takes 2-5 days to deliver results. In the meantime, patients are empirically treated with powerful, broad-spectrum antibiotics that may not be optimal or even effective at killing the pathogen causing the infection. Additionally, empirical treatment with broad-spectrum antibiotics contributes to the spread of antibiotic resistance.

[0417] Current methods use non-specific detection methods, such as increased turbidity, to determine which antimicrobial agents inhibit the growth of the target pathogen in the microbial medium, requiring lengthy cell purification processes. When using non-specific measurements of cell replication, one can only know that observed growth is due to the target pathogen if it contains only cells of the target pathogen. Cell purification must be performed for current antimicrobial susceptibility testing methods because the majority of medical specimens are not sterile. The specimens generally contain microorganisms that make up the human microbiome, the population of benign, normal bacteria that live in our bodies.

[0418] In contrast, the method of the present invention can deliver accurate antimicrobial susceptibility test results directly from the specimen without a colony purification step. This method differs from current methods in that it specifically assesses growth against the target pathogen in a microbial medium containing an antimicrobial agent.

[0419] In this example, we demonstrate that a 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 using the new method, antimicrobial susceptibility testing results are accurate and not significantly affected by off-target bacteria in urine samples that contain high concentrations of other microbial species. Experimental procedure.

[0420] 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 at 10x the desired concentration. The concentrations selected for testing of each antibiotic straddled the CLSI reported breakpoints for the antibiotic against E. coli. In addition to the wells containing the antimicrobial dilution series, the plate included four wells containing water to allow for positive and negative controls.

[0421] Culture preparation: 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 Three to five colonies of each of K. pneumoniae ATCC 23348-BAA 949, K. pneumoniae CDC 0043, and K. pneumoniae CDC 0141 were separately inoculated into 5 mL of Tryptic Soy Broth (TSB, Hardy Diagnostics catalog U65) and incubated at 35°C for 1 to 2 hours with shaking. The optical density was measured by spectrophotometer, and the organisms were diluted into 1× cation-adjusted Mueller-Hinton Broth (MHBII, Teknova catalog M5860). E. coli was diluted to approximately 5×10 6 CFU / mL (final assay concentration is 5 × 10 5 CFU / m), while other off-target species were diluted to various inocula (1 × 10 5 CFU / mL to 5×10 8 CFU / mL).

[0422] Preparation of magnetic particles: 2-hydroxypropyltrimethylammonium chloride coated silica magnetic particles (SiMag-Q, Chemicell, catalog 1206-5) were diluted to 2.75 × 10 in 50 mM EPPS buffer, pH 8.2. 12 Fluorescent magnetic microspheres containing a green dye (Dragon Green Fluorescent Microspheres, BANGS Laboratories, catalog MEDG001) were added to the suspension at a final concentration of 3 × 10 6 The magnetic particles were added at 1000x10000 particles / mL. These particles allow the optics to focus at the correct plane. The magnetic particle mixture was sonicated for 1 minute immediately before use to minimize clumping. Separate magnetic particle suspensions were prepared for the zero and 4 hour assays as described below.

[0423] Bacterial cell labeling at AST time zero: The assay signal before the onset of bacterial growth (time zero or T0) in the presence or absence of antibiotics was determined for each species of E. coli. 10 μL of each sample was added to 80 μL of hybridization buffer (3×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), 1×Cation-Adjusted Mueller-Hinton Broth (MHBII), E. coli specific DNA oligonucleotide FISH probes and unlabeled DNA helper probes). The probe sequences are shown in the table in Figure 78. 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. At this point, 100 μL of sample containing hybridization mixture, urine, and magnetic particles was transferred to a microtiter plate containing 50 μL per well of (pre-dried) "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 no. 3222)) and incubated at 35° C. for 30 minutes. After incubation, the microtiter plate was placed on a magnetic field (Dexter magnetic technologies, catalog 54170260) for 4 minutes to guide the magnetic particles, the fraction containing the labeled cells, through the "dye cushion" and near the imaging surface at the bottom of the well.

[0424] 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. The instrument uses a high-precision linear stage from Prior Scientific (Rockland, MA) to position each well above 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 fluorescent 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-bit quantization per pixel. A final image for each well is then formed by summing multiple frames. Sixteen frames were captured with a 100-millisecond exposure using a 635 / 25-nm excitation and 667 / 30-nm emission filter. Imaging of focal particles is performed with 470 / 40 nm excitation and 520 / 40 nm excitation filters, and 2 frames are captured with a 20 ms exposure.

[0425] 4 hours of growth: E. coli BAA 2469 were 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 were 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. Simultaneously with cell quantification at time zero, either E. coli species (5 × 10 6 CFU / mL) 10 μL, off-target species (1 × 10 5 ~5×10 8 10 μL of 100 μL of 1000 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 an antibiotic plate already containing 10 μL of antibiotic. Samples were grown for 4 hours at 35° C. in a static air incubator.

[0426] Bacterial cell labeling during AST at 4 hours of growth: After samples were incubated with and without antibiotics for 4 hours (T4), cells were labeled and quantified to determine how much, if any, growth had occurred. 10 μL (10%) of the incubated sample-antibiotic plate was transferred to a microtiter plate containing a dry "dye cushion" as described above for the assay at time zero and combined with 100 μL of a mixture of hybridization buffer, FISH probes, helper probes, magnetic particles, and focusing particles.

[0427] Comparison Method: The results of the novel assay method described herein were compared to broth microdilution (BMD) performed according to M07-Ed13E 2018.

[0428] Data analysis and threshold generation: Using the images captured by the CCD camera, the detected cells were estimated by an algorithm that looked at both the number of objects in the field of view and the intensity of the objects. The number of cells based on this detection algorithm was generated at zero time, as well as at 4 hours without antibiotic and at 4 hours with each antibiotic at all concentrations. For each bacterium sample / drug concentration, the growth fold was calculated as the signal in the well containing the antibiotic after growth (4 hours) relative to the signal in the urine sample before growth (zero time). Using the growth fold and the observation of growth in the corresponding well in CLSI-compliant broth microdilution, a logistic regression model was used to generate a threshold value to determine the growth fold cutoff above which the cells grew in the presence of antibiotic (and thus were resistant at that concentration) and below which the cells were in the process of death (and thus were sensitive at that concentration). The point at which the number of growth folds fell below the determined threshold is the MIC value generated by the assay. The results were assigned to categories of sensitive, intermediate, or resistant to each antibiotic accordingly. result.

[0429] The data presented demonstrates that the 4 hour AST method is robust to non-sterile samples, whereas the CLSI BMD method is not robust in the presence of excess bacteria.

[0430] Figure 73 shows data for E. coli BAA-2469 in the presence of nitrofurantoin and with increasing concentrations of S. aureus ATCC 25923 up to a 100-fold excess. The E. coli MIC in the CLSI-like broth microdilution method is affected by the addition of S. aureus strains (X in the figure), with the MIC increasing from 8 in the absence of S. aureus to 32 with a 100-fold excess of S. aureus. In contrast, the MIC for the novel 4-hour AST assay described in this invention (MultiPath, circles) was the same regardless of the amount of S. aureus cells (8) (dashed line).

[0431] Figures 75-77 show raw MIC values ​​determined using this novel method (MultiPath) compared to CLSI broth microdilution in the presence of E. coli BAA-2469 alone. The table in Figure 74 shows the overall essential agreement for E. coli in the presence of increasing off-target bacteria. Only a single condition - 1e7 Citrobacter freundii with nitrofurantoin - lacked essential agreement, but this did not change the determination of susceptible / intermediate / resistant categories, which were 100% concordant across all antibiotics and all off-target bacteria.

[0432] Conclusion: This example demonstrates that when the present invention is used for antimicrobial susceptibility testing, cell purification is not required to achieve accurate antimicrobial susceptibility test results for the target pathogen, even in samples containing many other species of other microorganisms.

[0433] Variations: This example illustrates the performance of this novel FISH method and is not limited to the specific details contained herein. Thus, those skilled in the art will 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, component concentrations), urine concentrations and urine processing procedures. This methodology can also be clearly extended to other biological specimens and other bacterial and non-bacterial pathogens.

[0434] FIG. 73 shows that the E. coli MICs were consistent by the above method with various inoculations of S. aureus, but the MICs by BMD increased with increasing S. aureus.

[0435] FIG. 74 shows a summary of the agreement between E. coli and standard BMD for off-target microorganisms at various inoculum levels.

[0436] FIG. 75 shows agreement with standard BMD for E. coli and off-target microorganisms (S. aureus, Staphylococcus epidermidis, and Citrobacter freundii) at various inoculum levels.

[0437] FIG. 76 shows agreement with standard BMD for E. coli and off-target microorganisms (Micrococcus luteus, Acinetobacter baumannii, Corynebacterium minutissimum) at various inoculum levels.

[0438] FIG. 77 shows the agreement with standard BMD for E. coli and an off-target microorganism (K. pneumoniae) at various inoculum levels.

[0439] FIG. 78 is a table of the probe sequences used in this Example 10.

[0440] Example 11 Rapid antimicrobial susceptibility testing is accurate for lactam antibiotics in the presence of beta-lactamase-expressing bacteria

[0441] Overview: Current methods of 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, takes 2-5 days to deliver results. In the meantime, patients are empirically treated with powerful, broad-spectrum antibiotics that may not be optimal or even effective at killing the pathogen causing the infection. Additionally, empirical treatment with broad-spectrum antibiotics contributes to the spread of antibiotic resistance.

[0442] One of the reasons current methods require lengthy cell purification processes is that they use non-specific detection methods, such as increased turbidity, to determine which antimicrobial agents inhibit the growth of the target pathogen in a microbial medium. When using a non-specific measurement of cell replication, one can only know that any growth observed is due to the target pathogen if it contains only cells of the target pathogen.

[0443] In contrast, the method of the present invention can deliver accurate antimicrobial susceptibility test results directly from the specimen without a colony purification step. This method differs from current methods in that it specifically evaluates growth against the target pathogen in a microbial medium containing an antimicrobial agent. In another example, the method of the present invention is demonstrated to be accurate in the presence of a large number of cells from off-target species.

[0444] This example addresses 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 delivers accurate antimicrobial susceptibility test results for target pathogens in artificial urine specimens that contain a large number of off-target species that produce enzymes known to degrade the antimicrobial agent being tested. In theory, this could potentially cause the concentration of the antimicrobial agent to change significantly enough to alter the antimicrobial susceptibility test results.

[0445] This example demonstrates that rapid antimicrobial susceptibility testing provides accurate antimicrobial susceptibility test results for two carbapenem antibiotics, meropenem and imipenem, even in the presence of numerous off-target pathogens that produce enzymes that destroy this type of antimicrobial.

[0446] Experimental Procedures. Antibiotic Plate Preparation: Antibiotic plates were prepared as described in the Impact of Non-Sterile Sample on Target MIC example.

[0447] Preparation of cultures: Three to five colonies each of E. coli ATCC 25922, a strain of bacteria sensitive to most antibiotics, and K. pneumoniae CDC 0141, a strain expressing beta-lactase 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 to 2 hours with shaking. Optical density was measured by spectrophotometry and organisms were diluted into 1× cation-adjusted Mueller-Hinton Broth (MHBII, Teknova catalog M5860). E. coli was diluted at 5×10 5 CFU / mL (CLSI standard concentration), whereas K. pneumoniae was diluted to various inocula (1 × 10 6 CFU / mL to 5×10 8 CFU / mL).

[0448] Preparation of magnetic particles: 2-hydroxypropyltrimethylammonium chloride coated silica magnetic particles (SiMag-Q, Chemicell, catalog 1206-5) were diluted to 3.75 × 10 in 50 mM EPPS buffer, pH 8.2. 6Fluorescent magnetic microspheres containing a green dye (Dragon Green Fluorescent Microspheres, BANGS Laboratories, catalog MEDG001) were added to the suspension at a final concentration of 3 × 10 6 The magnetic particles were added at 1000x10000 particles / mL. These particles allow the optics to focus at the correct plane. The magnetic particle mixture was sonicated for 1 minute immediately before use to minimize clumping. Separate magnetic particle suspensions were prepared for the zero and 4 hour assays as described below.

[0449] Bacterial cell labeling at AST time zero: The assay signal at time zero (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 1× cation-adjusted Mueller-Hinton Broth (MHBII) containing species-specific Alexa647N-labeled DNA oligonucleotide FISH probes and unlabeled DNA helper probes. The probe sequences used are shown in Table A. 100 μL of the mixture was then added to a well of a microtiter plate containing dehydrated hybridization buffer (3×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 per well of (pre-dried) "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. After incubation, the microtiter plate was placed over a magnetic field (Dexter magnetic technologies, Cat. 54170260) for 4 minutes to guide the magnetic particles, the fraction containing the labeled cells, through the "dye cushion" and into the vicinity of the imaging surface at the bottom of the well.

[0450] 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.

[0451] Four hours of growth: E. coli was tested for susceptibility to two antimicrobial agents: imipenem and meropenem in the presence of various inocula of K. pneumoniae-OXA. Cell quantification at time zero was performed simultaneously with the addition of 10 μL (5 × 10 6 CFU / mL), K. pneumoniae, 10 μL (1 × 10 6 ~1×10 8 10 μL of 1000 CFU / mL) or medium (control), 10 μL of pooled urine, and 60 μL of MHB II (Teknova, catalog number 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.

[0452] Bacterial cell labeling during AST at 4 hours of growth: After samples were incubated with and without antibiotics for 4 hours (T4), cells were labeled and quantified to determine how much, if any, growth 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 assay at time zero.

[0453] Comparison Methods: MulitPath™ assay results were compared to broth microdilution (BMD) performed according to CLSI method M07-Ed13E 2018.

[0454] Data analysis and threshold generation: Using the image captured by the CCD camera, the detected cells were estimated by an algorithm that looked at both the number of objects in the field of view and the intensity of the objects. The number of cells based on this detection algorithm was generated at zero time, as well as at 4 hours without antibiotic and at 4 hours with each antibiotic at all six concentrations. For each urine sample / drug concentration, the growth fold was calculated as the signal in the well containing the antibiotic after growth (4 hours) relative to the signal in the urine sample before growth (zero time). Using the growth fold and the observation of growth in the corresponding well in CLSI-compliant broth microdilution, a logistic regression model was used to generate a threshold value to determine the growth fold cutoff above which the cells grew in the presence of antibiotic (and thus were resistant at that concentration) and below which the cells were in the process of death (and thus were sensitive at that concentration). The point at which the number of growth folds fell below the determined threshold is the MIC value generated by this assay. The results were assigned to categories of sensitive, intermediate, or resistant to each antibiotic accordingly. All data were then compared to the CLSI standard BMD. Four hours of growth in the absence of antibiotics is the control condition to ensure that viable bacteria are present in the processed urine sample. result.

[0455] FIG. 79 shows the MICs of imipenem-susceptible E. coli strains 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 beta-lactamase producing K. pneumoniae, even at high concentrations, and the MICs are consistently less than 1 μg per mL of imipenem. In contrast, the BMD method after 16-24 hours of growth shows increased MICs for susceptible E. coli strains with increasing levels of K. pneumoniae, which would lead to them being erroneously determined to be resistant to this antibiotic.

[0456] Figure 80 shows similar results for the lactam antibiotic meropenem.

[0457] Conclusion: Our novel 4.5-hour AST method demonstrates accurate MIC determinations for bacteria susceptible to carbapenem antimicrobials, even in the presence of high concentrations of resistant bacteria expressing carbapenemase enzymes that degrade the antibiotics.

[0458] Variations. This example illustrates the performance of this novel AST method and is not limited to the specific details contained herein. Thus, those skilled in the art will 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, component concentrations, etc.), urine concentrations and urine processing procedures. This methodology can also be extended to additional combinations of lactam-susceptible and beta-lactamase-expressing bacteria.

[0459] FIG. 79 is a comparison of the new rapid AST method and the BMD method for determining imipenem MICs against E. coli in the presence of carbapenem-hydrolyzing B-lactamase-producing resistant strains of K. pneumoniae.

[0460] FIG. 80 shows that the E. coli MICs were consistent with various inoculations of carbapenem-hydrolyzing B-lactamase-producing resistant strains of K. pneumoniae using the above method, whereas the standard BMD was not.

[0461] FIG. 81 is a table of the probe sequences used in this Example 11.

[0462] Example 12 Accurate, rapid antimicrobial susceptibility testing of bacteria in urine without culture-based cell purification.

[0463] SUMMARY: Current methods of antimicrobial susceptibility testing require lengthy culture-based colony purification to ensure a pure population of pathogen cells, without the specimen itself. Thus, antimicrobial susceptibility test results, which indicate which antibiotic is best to kill the pathogen causing the infection, are not available for 2-5 days. In the meantime, patients are empirically treated with strong, broad-spectrum antibiotics that may not be optimal or even effective at killing the pathogen causing the infection. In addition, empirical treatment with broad-spectrum antibiotics contributes to the spread of antibiotic resistance.

[0464] In contrast, the method of the present invention can deliver accurate antimicrobial susceptibility test results directly from the specimen without lengthy colony purification steps. Here, we show that the new antimicrobial susceptibility test results are not significantly affected when bacteria in urine samples are tested without colony purification. In this example, we demonstrate that the rapid antimicrobial susceptibility test method accurately determines the minimum inhibitory concentration (MIC) for E. coli strains in artificial samples containing urine matrix (10%) for 15 different culture-negative urine samples.

[0465] Experimental Procedure. Urine Specimens: Fifteen culture-negative clinical urine samples (remainder) 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 presence of particles were noted. Upon receipt, urine was subjected to conventional urine culture to determine which samples were culture-negative. Briefly, a calibrated 1 μL loop was placed into a well-mixed urine sample and spread evenly on a tryptic soy agar (TSA) plate and incubated in a 35°C air incubator for 18-24 hours. The remaining urine samples were assayed as described below.

[0466] Antibiotic plate preparation: Microtiter plates were prepared containing six concentrations of each antibiotic in a two-fold serial dilution series starting at 10x the predicted minimum inhibitory concentration (MIC). The antibiotics used were cefazolin, ciprofloxacin, nitrofurantoin, and trimethoprim-sulfamethoxazole. Antibiotic dilutions were confirmed to fall within reasonable tolerability ranges, with MICs for at least two CLSI QC strains confirmed to fall within the QC ranges reported in CLSI document M100Ed29E-2019. The concentrations selected for testing of each antibiotic straddled the CLSI-reported breakpoints for the antibiotic against E. coli. Eight wells containing water or diluent were included on the plate in addition to the wells containing the antimicrobial dilution series to allow for positive and negative growth controls without antibiotic.

[0467] Culture preparation: E. coli (BAA-2469) logarithmic cultures were grown using 3-5 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 diluted to 5 x 10 in 1x cation-adjusted Mueller-Hinton Broth (MHBII, Teknova catalog M5860). 6 CFU / mL (final concentration, 5 × 10 in each 100 μL reaction) 5 CFU / mL).

[0468] 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 ...

[0469] Bacterial cell labeling at AST time zero: The assay signal before the onset of bacterial growth in the presence or absence of antibiotics (time zero or T0) was determined for each urine sample. 10 μL of diluted E. coli was added to 70 μL of hybridization buffer: final concentrations: 3× SSC (0.45 M NaCl, 0.045 M Na citrate) buffer (Sigma, Catalog No. S6639), 1% CHAPS (Sigma, Catalog No. C3023), 1% NOG (Sigma Catalog No. 08001), 1× cation-adjusted Mueller-Hinton Broth (MHBII) (from 2× stock) (Teknova, Catalog M5866), and non-specific DNA oligonucleotide FISH probes and unlabeled DNA helper probes (see Table A for probe labels, sequences, and concentrations). A final concentration of 10% urine was obtained by adding 10 μL of each individual urine directly to the mixture. 10 μL of the magnetic particle mixture prepared as described above was added directly to this mixture. At this point, 100 μL of the sample containing the hybridization mixture, urine, and magnetic particles was transferred to a microtiter plate containing 50 μL of (pre-dried) "dye cushion" per well (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 dryer at 60° C. for 3 hours, 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 to guide the magnetic particles, the fraction containing the labeled cells, through the "dye cushion" and into the vicinity of the imaging surface at the bottom of the well.

[0470] 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.

[0471] 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. Cell quantification was performed simultaneously with time zero, and 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.

[0472] 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.

[0473] Comparison Methods: Results for the MulitPath™ assay were compared to broth microdilution (BMD) performed according to CLSI method M07-Ed13E 2018.

[0474] 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. Results: The figure below shows that there is little or no matrix effect on the AST results.

[0475] Figure 82 shows the MICs for E. coli BAA-2469 determined by the new AST method (filled 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 zone of essential agreement.

[0476] Figure 83 summarizes the results obtained for all five antibiotics. 100% essential agreement and 100% categorical agreement to standard BMD was observed across 15 culture-negative clinical urine samples using the new AST method.

[0477] FIG. 84 shows the MICs determined using the new AST method compared to the MICs observed in the CLSI-compliant BMD process for 15 culture-negative clinical E. coli spiked urine samples across the five antibiotics tested.

[0478] FIG. 82 shows that for levofloxacin, the essential agreement with standard BMD for each of the 15 spiked culture-negative clinical UTI urine samples was 100%.

[0479] 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.

[0480] 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.

[0481] FIG. 82 shows the essential agreement across 15 urines.

[0482] FIG. 83 shows 100% essential agreement and 100% categorical agreement for each of the 15 spiked-in culture-negative clinical UTI urine samples against standard BMD. * Cefazolin, ciprofloxacin, levofloxacin, nitrofurantoin, and trimethoprim-sulfamethoxazole.

[0483] Figure 84 shows the MICs for 15 urine samples spiked with E. coli determined by the new AST method compared to the standard BMD method ("CLSI compliant"). Concentration (μg / mL).

[0484] FIG. 85 is a table of the probe sequences used in this Example 12.

[0485] Example 13 Rapid and accurate AST for multiple targets in multiple microorganisms

[0486] 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.

[0487] 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.

[0488] 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.

[0489] 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.

[0490] 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. 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 ...

[0491] 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.

[0492] 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.

[0493] 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.

[0494] 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.

[0495] Comparison Methods: Results using the novel AST method described herein were compared to broth microdilution (BMD) performed according to CLSI M07-Ed13E 2018.

[0496] 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. result.

[0497] Figures 152 and 153 summarize the results of all 48 different pairwise combinations with the antibiotic ciprofloxacin.

[0498] Figure 92 shows that all MICs determined for the target bacteria by the new 4.5 hour AST method were within the two-fold tolerable range observed for the gold standard BMD method for each target bacteria (determined in the absence of the second bacteria), regardless of the presence of a second susceptible or resistant bacteria (referred to as essential agreement).

[0499] FIG. 93 shows that the determination of susceptibility and resistance categories for each target bacterium by the new AST method was not affected by the pairwise combinations and was 100% concordant with the BMD determination.

[0500] 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.

[0501] 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.

[0502] FIG. 89 shows the ciprofloxacin-susceptible and -resistant strains used in this example.

[0503] FIG. 90 is the first half of a table of the probe sequences used in this Example 13.

[0504] FIG. 91 is the second half of a table of probe sequences used in this Example 13.

[0505] Figure 92 shows the essential agreement for polymicrobial infections with two target organisms. As can be seen below, the AST method described above gives an essential agreement of 100% against the standard BMD.

[0506] Figure 93 shows the category agreement for polymicrobial infections with two target organisms. As can be seen below, the AST method described above gives 100% category agreement to the standard BMD.

[0507] Example 14 Rapid and accurate detection of multiple target pathogens in a single specimen in an automated instrument cartridge

[0508] 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.

[0509] 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.

[0510] 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.

[0511] 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.

[0512] 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). 12The solution was diluted 1:20 to a concentration of 3 × 10 particles per mL. For this dilution, fluorescent magnetic microspheres containing a green dye (Dragon Green Fluorescent Microspheres, BANGS Laboratories, catalog MEDG001) were used at 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.

[0513] 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.

[0514] 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.

[0515] 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.

[0516] 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.

[0517] 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).

[0518] FIG. 94 shows a cartridge run in which E. coli / K. pneumoniae mixed samples were tested (N=10).

[0519] FIG. 95 shows a cartridge run in which E. coli / P. aeruginosa mixed samples were tested (N=10).

[0520] FIG. 96 shows cartridge runs where K. pneumoniae / P. aeruginosa mixed samples were tested (N=10). Cartridge number 6 for K. pneumoniae / P. aeruginosa was removed from the analysis because it 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, so the replicate of this signal was excluded. The replicate of this excluded 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 bacteria that were not spiked.

[0521] 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.

[0522] 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.

[0523] Figure 94 shows that the target pathogens are detected only in wells containing their species-specific DNA oligonucleotide FISH probes.

[0524] Figure 95 shows that the target pathogens are detected only in wells containing their species-specific DNA oligonucleotide FISH probes.

[0525] Figure 96 shows that the target pathogens are detected only in wells containing their species-specific DNA oligonucleotide FISH probes.

[0526] Figure 97 is a table "Table A of Example 14" which shows that the target pathogens were detected while other non-target pathogens were not detected.

[0527] FIG. 98 is a table, "Table B of Example 14," showing the probe sequences used in this Example 14.

[0528] (Example 16) Nonspecific detection of live bacteria using carboxy-fluorescein diacetate

[0529] Overview. In this example, large area imaging was used to detect individual S. aureus bacterial cell targets stained with a fluorogenic esterase substrate. This substrate can diffuse through the plasma membrane of intact live cells, both of which become fluorescent and charged when acted upon by esterase enzymes found in metabolically active cells. These charged fluorescent products can no longer passively diffuse through the plasma membrane and are trapped in the intact cell. Thus, this technique can distinguish live from dead cells, since only cells with active esterase and an intact plasma membrane are properly stained. In this example, S. aureus cells are labeled with the fluorogenic substrate carboxy-fluorescein diacetate (cFDA) and imaged using non-magnified digital imaging. Experimental method. Preparation of bacterial cells:

[0530] S. aureus ATCC 29213 was grown overnight in Tryptic Soy Broth (TSB, BD Cat. No. 211822). Logarithmic cultures of S. aureus were generated by inoculating 100 μL of the overnight culture into 5 ml of fresh TSB medium and further incubating at 35° C. in a shaking incubator for 2.5 hours. Preparation of magnetic particles:

[0531] Antibody-conjugated magnetic particles were made by coupling magnetic particles (Ademtec, 292 nm) to chicken anti-protein A antibody (Meridian Biosciences) using standard EDAC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) coupling chemistry. Labeling and capturing bacterial cells:

[0532] The assay was performed in a 96-well microtiter plate, where each well contained 40 μL of S. aureus (10,000 cells in TSB) or TSB only (no cells control), 5 μL of 10 mM cFDA (Life Technologies), and 5 μL of antibody-conjugated magnetic particles (2e10 / mL). The reactions were incubated at room temperature for 15 minutes. After incubation, 40 μL of the reaction mixture was carefully layered on top of 75 μL of a "dye cushion" (15% Optiprep with 5 mg / mL Chromotrope 2R) that had been pre-dispensed in a black, clear-bottom half-area microtiter plate (Greiner 675096, VWR part#82050-056). The microtiter plate was placed over a magnetic field for 4 minutes to guide the magnetic particles, the fraction containing the labeled cells, through the "dye cushion" and into the vicinity of the imaging surface at the bottom of the well. Imaging of bacterial cells:

[0533] After magnetic capture of the labeled cells:magnetic particle complexes, the microtiter plate was placed on a stage above a CCD digital camera (IDS, model UI-2250SE-M) and illuminated with light from an LED passing through an optical filter (469 nm, 35 nm FWHM). The fluorescent signal passing through an emission filter (520-35 nm) was detected by the camera to create an image of the fluorescent complexes. Images were analyzed using FLimage software (First Light Biosciences) to enumerate individual cells. result.

[0534] Figure 86 shows that S. aureus cells (left panel) are detected as bright fluorescent spots, while the medium without cells (right panel) contains only objects categorized as debris. The number of spots with labeled S. aureus cells in the field of view is approximately 5000, correlating well with the expected input bacterial count.

[0535] FIG. 87 shows S. aureus cells (left panel) and TSB medium only (right panel).

[0536] Conclusion. This example demonstrates a method for non-specific enumeration of bacteria. This technique can be used to count the total number of cells from a mixed population encompassing a wide range of bacterial species in a specimen. Specifically, this example demonstrates the ability of the non-magnification imaging method of the present invention to enumerate small numbers of bacterial cells labeled with carboxy-fluorescein diacetate, a fluorescent substrate that non-specifically labels viable cells that contain the ubiquitous esterase enzyme.

[0537] Variations. There are other stains for non-specific cell labeling, including STYO and SYBR stain families, propidium iodide, and nucleic acid stains such as DAPI. Other stains can be used that differentiate between live and dead cells. For example, other fluorogenic substrates that may or may not cross intact cell membranes, or DNA stains can be used instead of or in conjunction with cFDA or FDA. Multiple stains and dyes can be differentiated using multiple excitation and emission wavelengths for fluorescence detection. The spectrum of fluorescence associated with an object can be used to determine whether a cell is counted as a live cell or a dead cell. In addition, fluorogenic substrates specific for the biochemical activity of a particular type of bacteria can be used to determine the presence of that bacteria. For example, a fluorogenic β-galactosidase substrate can be cleaved into a fluorescent product by β-galactosidase that is specific for coliform bacteria. This methodology can also be applied to specimens that contain a large number of bacteria, and multiple species of bacteria. The method is suitable for detecting bacteria in many different clinical specimen types (eg, urine, sputum, swabs, spinal fluid, etc.) with minimal processing.

[0538] Example 15 Non-specific detection of bacteria using DNA staining dyes

[0539] Overview. In this example, wide-area imaging was used to detect individual live S. aureus bacterial cell targets stained with DNA-binding dyes. DNA-binding dyes can diffuse through the cell membrane of intact live cells, where they become highly fluorescent after binding to the bacterial DNA. Once bound to DNA, these dyes can no longer readily and passively diffuse through the intact cell membrane and become trapped within the intact cell. This technique can be useful when it is important to distinguish between live and dead cells, as live cells with intact cell membranes stain differently with different dyes compared to dead or membrane-compromised cells. In this example, S. aureus is labeled with SyBR Green, a DNA-binding fluorescent dye, and imaged using non-magnified wide-area CCD imaging. Experimental method. Preparation of bacterial cells:

[0540] S. aureus ATCC 29213 was grown overnight in Tryptic Soy Broth (TSB, BD Cat. No. 211822). S. aureus was grown to logarithmic growth by inoculating 100 uL of the overnight culture into 5 ml of fresh TSB medium and further incubated at 35° C. for 2.5 hours in a shaking incubator. Preparation of magnetic particles:

[0541] Antibody-conjugated magnetic particles were generated by coupling magnetic particles (Ademtec, 292 nm) to chicken anti-protein A antibody (Meridian Biosciences) using standard EDAC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) coupling chemistry. Labeling and capturing bacterial cells:

[0542] The assay was performed in a 96-well microtiter plate, where each well contained 40 μL of S. aureus (10,000 cells in TSB) or TSB only (no cells control), 5 μL of 500-fold diluted SyBR Green dye (Cat#S7563, Life Technologies) and 5 μl of antibody-conjugated magnetic particles (2e10 / mL). The reaction was incubated for 15 minutes at room temperature. After incubation, 40 μL of the reaction mixture was carefully layered on top of 75 μL of a "dye cushion" (15% Optiprep with 5 mg / mL Chromotrope 2R) that had been pre-dispensed in a black, clear-bottom half-area microtiter plate (Greiner 675096, VWR part#82050-056). The microtiter plate was placed over a magnetic field for 4 minutes to guide the magnetic particles, the fraction containing the labeled cells, through the "dye cushion" and into the vicinity of the imaging surface at the bottom of the well. Imaging of bacterial cells:

[0543] After magnetic capture of the labeled cell:magnetic particle complexes, the microtiter plate was placed on a stage above a CCD digital camera (IDS, model UI-2250SE-M) and illuminated with light from an LED through an optical filter (469 nm, FWHM of 35 nm). The fluorescent signal passing through an emission filter (520-35 nm) was detected by the camera and an image of the fluorescent complexes was created. FLimage software (First Imaging, Inc.) was used to enumerate individual cells. Images were analyzed using a Fluorescence Intensity Analyzer (Fluorescence Spectroscopy, Inc., Light Biosciences). result.

[0544] Figure 86 shows that S. aureus cells (left panel) are detected as bright fluorescent spots, while cell-free medium (right panel) contains only objects categorized as debris. The number of spots in the field containing labeled S. aureus cells is approximately 5000, correlating well with the predicted number of input bacteria.

[0545] FIG. 87 shows S. aureus cells (left panel) and TSB medium only (right panel).

[0546] Conclusions: This large-area, non-magnified imaging system is capable of detecting and enumerating bacterial cells labeled with SyBR Green, a DNA-binding dye that non-specifically labels live cells.

[0547] Variations. Other dyes that distinguish live or dead cells may be used. For example, other fluorescent DNA dyes that can or cannot pass through intact cell membranes can be used instead of or in conjunction with SyBR Green. Multiple dyes and pigments can be distinguished by using multiple excitation and absorption wavelengths for fluorescence detection. The fluorescence spectrum associated with the object can be used to determine whether the cells are counted as live or dead cells. In addition, fluorogenic substrates that are specific for the biochemical activity of a particular type of bacteria can be used to determine its presence. For example, a fluorogenic □-galactosidase substrate can be cleaved into its fluorescent product by □-galactosidase, which is specific for coliform bacteria. This methodology is also applicable to most bacteria and specimens containing multiple bacterial species. The method is suitable for detecting bacteria in many different clinical specimen types (e.g., urine, sputum, swabs, cerebrospinal fluid, etc.) with minimal processing. Other nucleic acid dyes, including other members of the SYTO / SYBR family of dyes, propidium iodide, and DAPI, can nonspecifically label bacterial cells.

[0548] (Example 17) Automated and highly sensitive detection of C. difficile toxin B in stool specimens using the system of the present invention

[0549] Overview: C. difficile causes more hospital-acquired infections and patient deaths than any other pathogen and is at the top of the CDC's list of imminent threats. Two of the current laboratory methods for diagnosing C. difficile infection are inaccurate. Enzyme immunoassay tests for C. difficile infection lack clinical sensitivity, i.e., they fail to detect patients with the disease. Nucleic acid amplification tests lack clinical specificity - these tests erroneously diagnose patients without the disease as positive for infection. Tests that are more sensitive to the C. difficile toxins that cause infection are both sensitive and specific. More accurate tests lead to better patient outcomes. This example demonstrates the use of the present invention to detect very low concentrations of C. difficile toxin B in stool samples. Experimental method.

[0550] Materials. Nanoparticles were attached to two monoclonal antibodies that bind to complementary epitopes on C. difficile toxin B protein. Fluorescent nanoparticles (Thermo Fisher Scientific, Waltham, MA) were conjugated with anti-C. difficile toxin B monoclonal antibody (BBI Solutions, Cardiff, UK). Carboxylated polystyrene magnetic particles (Ademtech, Pessac, France) were conjugated with anti-C. difficile toxin B monoclonal antibody (Fitzgerald, Acton, MA). Both fluorescent and magnetic particles were lyophilized after conjugation. Lyophilized particles are placed into First Light cartridges during assembly. Native toxin B protein purified from C. difficile was purchased from List Laboratories (Campbell, CA). Casein, casein hydrolysate, Trizma®-HCl were from Sigma-Aldrich (St. Louis, MO). Poly-BSA was from Roche. Protease inhibitor cocktail was from Takara Bio (Mountain View, CA). Spin columns were purchased from Pierce / Thermo-Fisher Scientific.

[0551] Estimation of the limit of detection for C. difficile toxin B testing on the MultiPath instrument. LoD measurements were performed using pooled negative stool samples. The limit of detection (LoD) was determined according to accepted Clinical & Laboratory Standards Institute (CLSI) guidelines by performing 24 replicates of samples without analyte and 12 replicates each with 5 different toxin B concentrations. Pooled negative stool samples were generated from 14 individual stool samples scored as C. difficile negative by real-time PCR. Pooled stool samples were spiked with C. difficile toxin B at a series of two-fold dilutions (0, 31.2, 62.5, 125, 250, 500 pg / mL). 100 μL of each stool sample was added to stool diluent (900 μL) consisting of Tris buffer, Poly-BSA, casein and protease inhibitor cocktail. 0.95 mL of each diluted sample was transferred to a Pierce spin column and centrifuged at 11,700 x g for 5 minutes. After centrifugation, 700 μL of the supernatant was transferred to the sample addition port of the cartridge and the cap was closed. The cartridge was then placed in the cartridge input rack and inserted into the instrument.

[0552] Running the cartridge on an automated instrument. After the cartridge was placed in the instrument, all subsequent actions were performed automatically, except for data analysis, which was performed offline using Excel or JMP software. The diluted stool samples were first directed under vacuum into individual reaction wells in the cartridge, transferred to the imaging window containing 46 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 no. 3222)), dried in a convection dryer at 60° C. for 3 hours, and incubated in the instrument at 35° C. for 30 minutes. After this incubation, the cartridge was then repositioned in the magnet station and placed on top of a strong permanent magnet (Dexter magnetic technologies, catalog 54170260) for 4 minutes to guide the fluorescent particle:toxin B:magnetic particle complexes through the "dye cushion" and into the vicinity of the imaging surface at the bottom of the well. Finally, the cartridge was transferred to the imaging station and images were acquired using a non-magnifying CCD imager as described below.

[0553] Instrument Imaging System and Imaging Process. The MultiPath 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. The system uses a custom-designed high-precision 3-axis positioning system 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 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-bit quantization per pixel. A final image for each well is then formed by summing multiple frames. For C. difficile toxin testing, the test channel is 470 / 40nm excitation and 520 / 40nm emission filters, and two frames are captured with a 20-millisecond exposure. Imaging of focal particles is performed with 569 / 25 nm excitation and 609 / 34 nm excitation filters, and 2 frames are captured with a 10 ms exposure. result.

[0554] Figure 99 shows that the method returned an estimated detection limit of 58 pg / mL for C. difficile toxin B. Signal variability across biological and technical replicates is shown by error bars (+ / - 1 standard deviation).

[0555] Conclusion: This method allows for highly sensitive and accurate detection of C. difficile toxin B. The LoD was determined to be 58 pg / mL toxin B. The low signal variability across technical and biological replicates indicates robustness to matrix effects.

[0556] Variations. This example illustrates the performance of the method of the present invention and is not limited to the specific details contained herein. Thus, it will be readily apparent to those skilled in the art that many variations are possible, including the use of different fluorescent particles, alternative assay chemistries (different buffers, pH, temperature, reaction time, component concentrations), different amounts of stool, and different stool sample processing means. Furthermore, alternative biomarkers specific for C. difficile can be used (e.g., toxin A). This novel technology can also be extended to other target molecules as well as various bacterial and non-bacterial pathogens for which specific biomarkers can be clearly identified. Incorporation by Reference

[0557] 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

[0558] 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) a transport system operable to move cartridges containing specimens to modules within the instrument; a magnetic module that attracts magnetic particles within the cartridge onto an imaging surface within the cartridge; an imaging module having a sensor that receives light from the imaging surface; and A control module that detects and counts the number of labeled molecules or cells bound to the magnetic particles. Including equipment. (Item 2) 2. The instrument of claim 1, wherein the control module and imaging module are capable of detecting and counting individual fluorescently labeled cells or molecules on the imaging surface without magnification. (Item 3) 2. The apparatus of claim 1, wherein the imaging module includes an XYZ stage operable to move the cartridge in the x, y, and z directions for positioning during imaging. (Item 4) 2. The instrument of claim 1, wherein the control module operates the XYZ stage to count individual cells in a series of different imaging wells within the cartridge. (Item 5) 2. The instrument of claim 1, wherein the imaging module includes an optical assembly mounted below a deck supporting the cartridge that covers the optical assembly, the optical assembly including one or more of the sensor, light source, lens, and filter wheel. (Item 6) 2. The instrument of claim 1, further comprising a fluidics module that uses pressure to split the analyte from an input well on the cartridge into a split well in the cartridge. (Item 7) 2. The instrument of claim 1, further comprising at least one heater block on said deck capable of maintaining the cartridge at a controlled temperature. (Item 8) 2. The device of claim 1, wherein the magnetic module has a magnet disposed on a bottom surface of the cartridge slot. (Item 9) 2. The apparatus of claim 1, wherein the mechanical transport system includes a carousel and a mechanical arm that moves cartridges between the carousel and the module. (Item 10) 2. The instrument of claim 1, wherein the control module reads a code from the cartridge and analyzes the counted labeled molecules or cells to provide a test result corresponding to the code. (Item 11) 11. The device according to item 10, wherein the test is a microbial identification test. (Item 12) 11. The instrument of claim 10, wherein the test is an antimicrobial susceptibility test and the control module analyzes differential growth between wells of the cartridge. (Item 13) 13. The device of item 12, wherein the cartridge comprises a plurality of wells containing different antimicrobial agents or different concentrations of one or more antimicrobial agents. (Item 14) 2. The instrument of claim 1, further comprising an incubation station operable to maintain the contents of the cartridge at a desired incubation temperature when the cartridge is placed therein. (Item 15) 2. The instrument of claim 1, further comprising a disposal module for disposing of the cartridge after performance of the testing step. (Item 16) 10. The instrument of claim 9, wherein the carousel is enclosed and the instrument is operable to maintain the carousel at an incubation temperature required to perform the testing steps. (Item 17) the carousel and the module include slots sized to accommodate the cartridges; Rotation aligns the carousel slots with the module slots; 10. The instrument of claim 9, wherein the mechanical arm is operable to push the cartridge between the carousel and the module slot. (Item 18) 2. The instrument of claim 1, wherein the control module comprises a processor coupled to a non-transitory tangible memory and operable to accept inputs specifying tests to be performed on the specimens in the cartridge and to control the instrument to perform the tests. (Item 19) Conducting the test comprises: accessing a test step routine from said non-transitory tangible memory corresponding to said test being performed; accessing existing scheduled routines for already performed cartridge tests within said instrument; scheduling the test step routines for the tests to be performed on the cartridge to avoid conflicts with the existing scheduled routines; and executing said test step routine on said cartridge within said instrument by rotating said carousel and moving said cartridge among one or more of a plurality of stations corresponding to said test step routine; Item 19. The device according to item 18, comprising: (Item 20) 20. The instrument of claim 18, wherein an input designation specifying the test to be performed is received from a scanning device in communication with the processor and operable to read a tag on the cartridge indicating the test to be performed.

Claims

[Claim 1] The invention described in the specification.

Images