Test Cartridge

Abstract

The present disclosure provides cartridges preloaded with reagents for performing antimicrobial susceptibility testing (AST) and FISH testing. The cartridges of the present disclosure contain various incubation wells loaded with different antimicrobial agents for differential growth analysis. Imaging wells with species-specific microbial probes, fluorescent tags, magnetic particles, and a dye cushion layer enable tagging and imaging of target microorganisms for differential growth analysis. The present disclosure relates to systems, devices, and methods useful for detecting infections, identifying infectious pathogens, and determining effective antimicrobial treatments for infections.

Description

[Technical field]

[0001] The present disclosure relates to systems, devices and methods useful for detecting infections, identifying infectious pathogens, and determining effective antimicrobial treatments for infections. [Background technology]

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

[0003] Unfortunately, traditional methods used to identify effective targeted antibiotics, called antimicrobial susceptibility testing (AST) methods, require several days to obtain results. One reason traditional antimicrobial susceptibility tests take so long is that they require large numbers of purified pathogen cells - on the order of millions. Purifying that number of cells by culturing in petri dishes requires one or more days, using a colony purification method that is over 130 years old. Once the purified cells are available, it takes one or more days to identify the pathogen and determine which antibiotics are effective to treat the patient.

[0004] On the one hand, patients are "empirically" treated with broad-spectrum antibiotics that kill a wide range of pathogens that may cause infection. Although these drugs can treat a wide range of pathogens, they are generally not the optimal treatment for the patient's specific pathogen and may not effectively treat the infection. The empirical use of broad-spectrum antibiotics also causes the spread of antibiotic resistance. These broad-acting drugs cause resistance not only in disease-causing pathogens but also in the trillions of benign microorganisms that reside in the human body. Further exacerbating the spread of antibiotic resistance is the fact that, in the absence of rapid diagnostic methods to determine which patients actually have an infection, uninfected patients are frequently unnecessarily treated with resistance-causing antibiotics.

[0005] Prompt determination of effective antimicrobial treatment can not only improve medical outcomes but also lower healthcare costs. For example, common life-threatening hospital-acquired infections, such as surgical site infections and ventilator-associated pneumonia, are responsible for nearly $10 billion in healthcare costs in the United States. Length of hospital stay is the largest cost attributable to these infections. Treating patients with optimal antimicrobial therapy close to the onset of symptoms can significantly accelerate their recovery and reduce lengthy and costly hospital stays. Summary of the Invention [Means for solving the problem]

[0006] Rapid and accurate identification of patients with infections and rapid implementation of effective treatments for these patients can save lives and slow the spread of antimicrobial resistance. The present invention provides a cartridge device that can accurately identify patients with infections in about 30 minutes and determine targeted treatments for the patient's infections in hours, compared to the several days required by today's methods. By detecting infections at a stage much closer to the onset of symptoms and identifying effective targeted antimicrobial agents, the present invention can dramatically improve medical outcomes and minimize empirical treatment with resistance-causing broad-spectrum antibiotics.

[0007] Cartridges according to the invention can be used to eliminate the time-consuming steps required by conventional methods to generate large numbers of purified cells. The cartridges can be preloaded with reagents for performing antimicrobial susceptibility testing (AST).

[0008] 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.

[0009] 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).

[0010] 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.

[0011] After detecting infection and identifying and quantifying the infectious pathogen, the cartridge invention and related inventive methods can determine which patient treatments will be most effective. This type of analysis is called antimicrobial susceptibility testing (AST). The present invention differs from current methods for antimicrobial susceptibility testing in that it can obtain accurate results directly from a patient specimen in just a few hours, instead of the current conventional methods that take days. Conventional methods, unlike the inventive method, 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 a patient specimen without time-consuming culture steps.

[0012] The novel and potentially medically impactful performance and utility of the present invention is made possible by the cartridge of the present invention and the associated system and method of the present invention to enable single molecule counting and single cell counting using non-magnified digital imaging of informative biological targets directly from patient specimens. The use of complex and expensive microscopes and simple, low-cost cameras without optics to digitally count microscopic cells and submicroscopic molecules allows for the detection of infections with rapid, sensitive and automated quantification of disease-causing toxins and disease-specific biomarkers. The system and method of the present invention for identifying and digitally counting pathogen cells underpins the ability to rapidly determine susceptibility or resistance to antimicrobial agents. The method of the present invention determines whether a pathogen is susceptible to an antimicrobial agent by determining whether the agent stops normal pathogen growth (i.e., an increase in cell number through cell division) when incubated in a microbiological nutrient medium. This can be performed in the cartridge device of the present invention by counting pathogen cells before and after incubation in a medium containing an antimicrobial drug.

[0013] The cartridges can be operable with instruments that automatically and simultaneously perform a variety of tests that require only sample input and provide actionable results at a variety of sites ranging from point of care to centralized hospitals and reference laboratories. Such testing is made possible by the combination of application-specific cartridges preloaded with all necessary reagents to minimize performance time for the user, sample input directly into the cartridge and full automation of all processing and analysis, and instruments designed for scalable throughput.

[0014] Cartridges of the invention, which may be operable by automated instruments to perform tests using methods for counting single molecules and single cells, may include fluorescently labeling target molecules or cells, magnetically tagging the targets, using magnetic forces to deposit the fluorescently labeled magnetic targets on an imaging surface of the device, imaging the targets without magnification (or with minimal magnification), and counting the targets using image analysis.

[0015] 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 instrument and cartridges of the present invention allows for operation by medical professionals without significant specialized training. Furthermore, the breadth of potential test menus for this instrument and application-specific cartridges designed to work with the instrument reduces benchtop space, allowing for more cost-efficient utilization of equipment, and offers the potential for near-patient diagnostic testing to provide clinicians with potentially life-saving diagnostic information closer to the onset of infection, when they may have the greatest impact.

[0016] Application-specific cartridges can be preloaded with test reagents. Preferably, cartridges can be assembled and packaged with the necessary test reagents during manufacture and can be dispensed with the user only needing 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, can be pre-enriched. For example, blood specimens can 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.

[0017] The cartridge may be preloaded with fluorescent probes and reagents for identifying and quantifying target cells or molecules. For example, to detect cells using FISH-based methods, the cartridge may contain preloaded reagents for permeabilizing target cells, hybridization reagents, fluorescent target-specific oligonucleotide probes, and target-binding magnetic particles. The AST cartridge may contain microbiological media and antimicrobial agents to promote differential growth, in addition to the reagents for FISH-based methods for quantifying target cells after differential growth.

[0018] In a preferred embodiment of the present invention, the cartridge is used for antimicrobial susceptibility testing, and the specimen is divided into separate portions containing nutrient growth medium to promote the replication or growth of microbiological cells. One or more of these portions can be used as a reference or baseline portion to be directly processed and analyzed before incubation at a temperature that promotes growth to determine the number and quality of pathogen cells. One or more of these portions can be incubated at a temperature that promotes the growth of pathogen cells to determine whether the pathogen cells are viable. Additionally, each of the other portions contains a specific concentration of one or more antimicrobial agents and is incubated to determine the effect of the antimicrobial agents on pathogen cell replication.

[0019] The cartridge may interface with an instrument operable to manipulate the specimen therein, incubate the cartridge, and perform the necessary processing and imaging steps, so that the user need only load the specimen into the cartridge and receive the results. The cartridge may contain a growth medium and antimicrobial agent specifically selected for the particular microorganism, so that if a particular infection such as E. coli is suspected, the user may select the appropriate cartridge preloaded with E. coli specific reagents (e.g., media, antimicrobials, and FISH probes). Through the combination of an AST specific cartridge preloaded with all the necessary reagents, an automated instrument with separate stations for performing the assay steps and random access thereto within the instrument, and computerized scheduling and manipulation of multiple cartridges and assays, the system and method of the present invention can automatically and simultaneously perform a variety of assays that require only specimen input and provide actionable results in a point-of-care environment. The automated nature of the cartridges and associated instruments allows for operation by medical professionals without significant specialized training.

[0020] Cartridges can be set up with the necessary reagents during manufacture and distributed to point-of-care facilities where the user only needs to add the specimen to be tested (e.g., a blood specimen from a patient) and insert the cartridge into the instrument. The instruments described herein use a variety of different stations for performing different assay steps, positioned around a carousel that is used to receive, store, and move the assay-specific cartridges between the stations according to the assay being performed.

[0021] The cartridge may include a specimen chamber for receiving the specimen, and several separate or incubation wells preloaded with different antimicrobial agents. The cartridge may also include multiple imaging wells, each imaging well corresponding to a single separate well. The imaging wells may contain the necessary reagents to treat and image the target microorganism after incubation in the presence of various agents to provide its differential growth analysis and determine the most effective treatment for a patient's infection with a particular target microorganism.

[0022] The specimen chambers, the split or incubation wells, and the imaging wells may be connected to each other via a series of channels and valves, such as a sliding bar disposed between the split wells and the imaging wells. The bar may include vertical channels and be horizontally slidable so that the channels can align with the outlet channels of the split wells and the inlet channels of the corresponding imaging wells to create a fluidic path between them. When slid horizontally in either direction, the channels of the bar may not align with the wells, thereby isolating the wells and preventing fluidic communication therebetween. The valves may be externally manipulated by the instruments described herein depending on the assay steps to be performed.

[0023] The cartridge may include a pneumatic or other interface for connection to a pneumatic source to apply a pressure gradient within the cartridge to move the analyte between the various compartments. When manipulated in conjunction with the valves, the pneumatic source may be used to open channels between the various compartments within the cartridge and then direct the analyte fluid through the open channels.

[0024] The cartridge of the present invention may be operable by the instruments described herein using a variety of different stations for performing different test steps on the cartridge device of the present invention as outlined above. The stations may be positioned around a carousel that is used to receive, store, and move the application-specific cartridge 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.

[0025] In a preferred embodiment, the test uses a constant or cyclic temperature throughout all steps, or is modified so that the interior of the instrument can be maintained at the required temperature and the carousel can act as a storage station for incubation steps. The temperature can be physiological, or below or above physiological temperature.

[0026] The instrument that operates the cartridge can use a carousel to access different stations so that the test steps can be performed in the order and timing required for the various types of tests. Precise computer scheduling and computer-controlled access to the various stations in the instrument is used to automatically perform all steps of the various tests without further user input. After loading the cartridge into the instrument, the user's next interaction 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.

[0027] The cartridges may be labeled with one or more bar codes or other identifiers that may be read by a human or automatically read by an automated instrument to associate patient, test application specific or factory information with the cartridge. The instrument may also use its inputs to record and track information associated with the specimens being tested, including patient information, for reporting results. The instrument may also use its inputs to record and track information associated with the specimens being tested, including patient information, for reporting results.

[0028] An instrument for processing cartridges of the present invention may include a computer including a processor and non-transitory tangible memory, operable to schedule and control tests being performed in the instrument and to track cartridges therein. 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.

[0029] Instruments designed to manipulate cartridges may include mechanical conveyor arms to move cartridges between the carousel and various stations for performance of required test steps. In a preferred embodiment, the carousel and stations include slots sized to receive and position the cartridges within the stations. Rotation of the carousel may align the carousel slots with corresponding slots in the appropriate stations, and the mechanical conveyor arms may be operable to contact the sides of the cartridge and slide the cartridge along the aligned slot into the selected station. The mechanical conveyor arms avoid gripping the cartridge, reducing jamming associated with gripping mechanisms. The mechanical conveyor arms 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 gripping mechanisms to move the cartridge.

[0030] Preloaded cartridges can allow control of reagent volumes and dispensing at the manufacturing site, and automated equipment controls the performance of test steps and their timing. Thus, the systems and methods 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.

[0031] 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.

[0032] The cartridges can be designed to perform infection detection, target identification, and effective treatment determination directly from patient specimens, such as urine, sputum or other respiratory specimens, blood, feces, wound specimens, or cerebrospinal fluid, with little or no specimen preparation steps. For example, urine specimens are pipetted directly into the cartridge 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 cartridge 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 cartridge 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.

[0033] The cartridge of the present invention may allow for direct processing and imaging of a specimen in the cartridge device of the present invention to determine the presence and identity of target cells present in the specimen. As described above, the processing and imaging steps may occur with the specimen in the cartridge test device, which may require little to no specimen preparation outside the cartridge. By discarding time-consuming specimen preparation techniques and using target-specific identifiable labels, the system and method of the present invention allows for the identification and enumeration of targets in a specimen in as little as 30 minutes or less.

[0034] The cartridge can be used to identify pathogens causing infection. For example, the cartridge of the present invention can be used to directly identify and quantify pathogens in patient specimens without the need for culture-based microbiological pre-enrichment or nucleic acid amplification. A preferred method enumerates target pathogen(s) in a single reaction mixture by labeling using a fluorescent in situ hybridization (FISH)-based method combined with magnetic selection, which can be carried out in about 30 minutes in a microtiter plate or cartridge in the device described herein.

[0035] The cartridges 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 microbial pathogens in a patient specimen. This information provides the clinician with information about which antimicrobial should be used to effectively treat that particular patient's infection.

[0036] 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.

[0037] 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.

[0038] Both the traditional method for antimicrobial susceptibility testing and the method that can be used for the more rapid testing in the cartridge of the present invention follow the steps described above, but the method that can be enabled by the present invention determines the antimicrobial susceptibility profile of a pathogen in a few hours, whereas the traditional method requires several days. The rapid antimicrobial susceptibility test results using the method of the present invention arise from the ability of the new method to test patient specimens directly 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.

[0039] 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.

[0040] Several novel aspects of the methods enabled by the cartridge of the present invention allow for rapid acquisition of antimicrobial susceptibility results directly from patient specimens.

[0041] First, patient specimens generally contain several orders of magnitude fewer cells than are required for conventional antimicrobial susceptibility testing. In contrast to current culture-pre-enrichment dependent methods, the cartridge can be engineered to 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 - after only a few generations of bacteria - determine whether cells have increased in number in an aliquot containing antimicrobial drug and growth medium.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] In contrast to conventional methods, such as those requiring purified pathogen cells due to the use of non-specific methods to detect whether a pathogen grows in the presence of an antimicrobial drug, the method deployed in the cartridge of the present invention uses pathogen-specific detection to evaluate the growth of the pathogen in the antimicrobial drug. Since only disease-causing pathogen cells are enumerated after the incubation step (and not any commensal microorganisms), the method of the present invention can be used to directly determine antimicrobial susceptibility in non-sterile primary specimens containing one or many commensal microbial species.

[0046] The cartridge device can be used to identify target cells or microorganisms and test for antimicrobial susceptibility of targets in a specimen, either separately or in the same cartridge. In a preferred embodiment of the present invention, the test device divides the specimen internally into separate portions, where some of these portions can be incubated in the presence of different antimicrobial agents before imaging to determine differential growth. One or more of these portions can be directly processed and imaged to provide a baseline reference for determining growth, growth inhibition or morphological changes in the incubated portions. By quantifying the growth of the portions 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.

[0047] The cartridges described herein and the instruments that operate them are capable of identifying and testing the effectiveness of agents against various classes of targets (e.g., viruses, human cells, bacterial cells or fungal cells), and also performing such identification and antimicrobial susceptibility testing simultaneously on multiple different targets in a single device, thereby enabling the development of a single instrument that performs tests typically performed by multiple testing devices designed for different testing applications (e.g., blood, urinary tract, gastrointestinal and respiratory infections). Thus, robust functionality is provided by the cartridges described herein and the instruments that operate them.

[0048] The cartridge of the present invention is used to rapidly obtain antimicrobial susceptibility results directly from a patient specimen. Patient specimens generally contain several orders of magnitude fewer cells than the number of cells required for conventional antimicrobial susceptibility testing. Using the cartridge of the present invention, small numbers of pathogen cells can be enumerated by sensitive single-cell counting using non-magnified digital imaging, in contrast to current culture-pre-enrichment dependent methods. Furthermore, because small numbers of individual cells are enumerated, the present invention can very quickly - within just a few generations of bacteria - determine whether cells have increased in number in an aliquot containing an antimicrobial drug and growth medium.

[0049] 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 those known to inhibit the growth of the identified target). As mentioned above, 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 cartridge 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 that are not within the scope of the present invention, such as amplification with target-specific primers, immunoassays, mass spectrometry, nucleic acid sequencing, or oligonucleotide probe array analysis. When identification is performed separately, the cartridge of the present invention may be used as an antimicrobial susceptibility testing device containing appropriate reagents, and antimicrobials against the identified pathogen may be used according to the present invention.

[0050] 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.

[0051] In certain embodiments, the test suite of the cartridge device may be used for infection detection as well as pathogen identification (ID) and antimicrobial susceptibility testing (AST) for symptomatic infections (e.g., pneumonia or urinary tract infections). Such test suites, referred to as ID / AST test suites, may include an ID cartridge and an infection-specific family of AST cartridges, each of which contains the appropriate antimicrobial for testing an individual pathogen or related group of pathogens. For example, a urinary tract infection (UTI) test suite may include a UTI ID cartridge and a family of UTI AST cartridges.

[0052] The ID cartridge in such an infection-specific test suite can detect infection and identify and quantify the bioburden or concentration of 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 on the same specimen, cartridge, and instrument is a novel and potentially powerful advantage of the systems and methods of the present invention.

[0053] 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.

[0054] Antimicrobial susceptibility test results obtained using the cartridge of the present invention are then used to determine the antimicrobial susceptibility profile for the infectious pathogen and to inform treatment decisions for the patient so that the patient can be treated with an effective antimicrobial agent.

[0055] Detectable labels incorporated into the cartridge of the present invention for specifically detecting and quantifying target cells may include target-specific fluorescent oligonucleotide probes (including probes containing modified nucleotides or nucleotide analogs), fluorescent antibodies, specific and non-specific ligands, lectins, dyes, or dyes that bind to targets. 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 to be 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, eliminate washing steps, and 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.

[0056] The cartridge of the present invention may be designed to perform 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 the cartridge device and divided into multiple aliquots. One aliquot is immediately analyzed to determine the 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 cartridge. 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.

[0057] 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 cartridge, 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.

[0058] For cartridges designed for antimicrobial susceptibility testing applications, target pathogen cells can be specifically detected after differential growth, as described above.

[0059] The cartridge of the present invention can use a similar strategy 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.

[0060] 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.

[0061] 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.

[0062] The cartridge and associated method may be operable to determine a recommended antimicrobial agent to treat the patient's infection, which typically includes an antimicrobial or other treatment determined to inhibit the growth of the target. The determination of the treatment that inhibits the growth of the target may occur several hours (e.g., 4 hours) after the specimen is introduced into the test device. The body specimen may be a tissue specimen (e.g., 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.

[0063] In certain embodiments, the cartridge of the present invention is operable to determine the therapeutic susceptibility of target cells or microorganisms in a specimen. Such a cartridge can receive a specimen containing a body fluid and target cells or microorganisms from a patient, and an instrument or instruments. 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.

[0064] The system may include a cartridge 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.

[0065] The cartridge may be operable by the instrument 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 between the target, magnetic tag and detectable label is specifically formed; apply a magnetic field to the test device to attract the complex to the detection surface; image the detection surface; and perform image analysis to determine the test results.

[0066] In the cartridges of the present invention, detecting the number of identified target cells or microorganisms in each of the incubated specimens may include contacting the incubated specimens 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.

[0067] 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).

[0068] The cartridge according to the present invention can be used to eliminate the time-consuming steps required by conventional methods to generate large numbers of purified cells. The cartridge is preloaded with reagents for performing antimicrobial susceptibility testing (AST) and FISH testing. The cartridge of the present invention is used to detect infections and identify infectious pathogens and effective antimicrobial agents in hours rather than days required by conventional methods. By detecting infections at a stage much closer to the onset of symptoms and identifying effective targeted antimicrobial agents, the present invention can dramatically improve medical outcomes and minimize empirical treatment with resistance-causing broad-spectrum antibiotics.

[0069] In particular, the cartridge of the present invention is used to rapidly obtain antimicrobial susceptibility results directly from a patient specimen. Patient specimens generally contain several orders of magnitude fewer cells than the number of cells required for conventional antimicrobial susceptibility testing. Using the cartridge of the present invention, small numbers of pathogen cells can be enumerated by sensitive single cell counting using non-magnified digital imaging, in contrast to current culture-pre-enrichment dependent methods. Furthermore, because small numbers of individual cells are enumerated, the present invention can very quickly - within just a few generations of bacteria - determine whether cells have increased in number in an aliquot containing an antimicrobial drug and growth medium.

[0070] In addition, patient specimens contain sample matrix and commensal microorganisms unrelated to infectious pathogens. Guidelines for conventional methods, such as those from the Clinical Laboratories Standards Institute (CLSI) or the European Committee on Antimicrobial Susceptibility Testing (ECAST), require purified cultured 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.

[0071] 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. This is an important reason underlying why conventional and most emerging antimicrobial susceptibility testing methods require pure cultures of cells. To determine antimicrobial susceptibility profiles, conventional and most emerging methods evaluate the effect of different antimicrobials at different concentrations on the growth of the target pathogen. The reason these methods require pure populations 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 aliquots, it is not possible to say using a general method for detecting growth whether the disease-causing pathogen, or one or more of the commensal species, were resistant and capable of growing.

[0072] In contrast to conventional methods, such as those requiring purified pathogen cells due to the use of non-specific methods to detect whether a pathogen grows in the presence of an antimicrobial drug, the present invention uses pathogen-specific detection to evaluate the growth of a pathogen in the presence of various antimicrobial drugs. Because only disease-causing pathogen cells are enumerated after the incubation step (and not any commensal microorganisms), the cartridge of the present invention can be used to directly determine antimicrobial susceptibility in non-sterile primary specimens containing one or many commensal microbial species.

[0073] Cartridges according to the invention can be used for pathogen identification to determine whether a specimen contains a sufficient number of cells of a pathogen species suspected of causing an infection. Cartridges according to the invention can be used for antimicrobial susceptibility testing to determine which of one or more antimicrobial agents are capable of preventing normal cellular replication of a pathogen suspected of causing an infection in a patient specimen. Such antimicrobial agents can potentially be used to effectively treat an infection in a patient.

[0074] The cartridge contains various division or incubation wells in which growth medium and various antimicrobial agents can be preloaded so that a single specimen can be added to the cartridge and the target microorganisms present in the specimen can be divided among the wells to allow incubation in the presence of various agents. After incubation, the specimen can be processed and imaged, for example, using fluorescent in situ hybridization (FISH), to provide differential growth analysis to determine the susceptibility of the target microorganism to various antimicrobial agents. AST can use FISH at a constant physiological temperature. Using the FISH protocol, the cartridge is useful for microbial identification testing or antibiotic susceptibility testing. Clinical specimens studied using the cartridge of the present disclosure are incubated and testing is performed at a temperature that matches the temperature of the patient, so that the relevant infectious bacteria growing in the patient continue to grow in the cartridge.

[0075] The cartridge is preloaded with fluorescent probes and reagents that chemically permeabilize microorganisms so that pathogenic bacteria can be fluorescently labeled and imaged without heating significantly above body temperature. Because the FISH protocol is performed at physiological temperatures, clinical specimens are not exposed to extreme heat that promotes clinically misleading bacterial growth patterns. The cartridge is designed for microbial identification or susceptibility testing directly from patient specimens, e.g., feces or urine, without the need for any specimen preparation. The cartridge and associated reader instrument provide test results within hours, giving clinicians the ability to quickly and easily identify the cause of an infection or which antibiotic treatments will be effective.

[0076] In a preferred embodiment of the present invention, the cartridge is used for antimicrobial susceptibility testing, and the specimen is divided into separate portions containing a nutrient growth medium that promotes the replication or growth of microbiological cells. One or more of these portions can be used as a reference or baseline portion that is directly processed and analyzed before incubation at a temperature that promotes growth to determine the number and quality of pathogen cells. One or more of these portions can be incubated at a temperature that promotes the growth of pathogen cells to determine whether the pathogen cells are viable. Additionally, each of the other portions contains a specific concentration of one or more antimicrobial agents and is incubated to determine the effect of the antimicrobial agents on pathogen cell replication.

[0077] The cartridge may interface with an instrument operable to manipulate the specimen therein, incubate the cartridge, and perform the necessary processing and imaging steps, so that the user need only load the specimen into the cartridge and receive the results. The cartridge may contain a growth medium and antimicrobial agent specifically selected for the particular microorganism, so that if a particular infection such as E. coli is suspected, the user may select the appropriate cartridge preloaded with E. coli specific reagents (e.g., media, antimicrobials, and FISH probes). Through the combination of an AST specific cartridge preloaded with all the necessary reagents, an automated instrument with separate stations for performing the assay steps and random access thereto within the instrument, and computerized scheduling and manipulation of multiple cartridges and assays, the system and method of the present invention can automatically and simultaneously perform a variety of assays that require only specimen input and provide actionable results in a point-of-care environment. The automated nature of the cartridges and associated instruments allows for operation by medical professionals without significant specialized training.

[0078] Cartridges can be set up with the necessary reagents during manufacture and distributed to point-of-care facilities where the user only needs to add the specimen to be tested (e.g., a blood specimen from a patient) and insert the cartridge into the instrument. The instruments described herein use a variety of different stations for performing different assay steps, positioned around a carousel that is used to receive, store, and move the assay-specific cartridges between the stations according to the assay being performed.

[0079] The cartridge may include a specimen chamber for receiving the specimen, and several separate or incubation wells preloaded with different antimicrobial agents. The cartridge may also include multiple imaging wells, each imaging well corresponding to a single separate well. The imaging wells may contain the necessary reagents to treat and image the target microorganism after incubation in the presence of various agents to provide its differential growth analysis and determine the most effective treatment for a patient's infection with a particular target microorganism.

[0080] The specimen chambers, the split wells and the imaging wells can be selectively connected to each other via a series of channels and valves, such as a sliding bar disposed between the split wells and the imaging wells. The bar can include vertical channels and be horizontally slidable so that the channels can align with the outlet channels of the split wells and the inlet channels of the corresponding imaging wells to create a fluidic path between them. When slid horizontally in either direction, the channels of the bar can be out of alignment with the wells, thereby isolating the wells and preventing fluidic communication therebetween. The valves can be externally manipulated by the instruments described herein depending on the assay steps to be performed.

[0081] The cartridge may include a pneumatic or other interface for connection to a pneumatic source to apply a pressure gradient within the cartridge to move the analyte between the various compartments. When manipulated in conjunction with the valves, the pneumatic source may be used to open channels between the various compartments within the cartridge and then direct the analyte fluid through the open channels.

[0082] The instrument for receiving and manipulating the cartridges can use a carousel to randomly access different stations so that multiple tests can be performed. Accurate computer scheduling and computer-controlled random access to various stations in the instrument is used to automatically perform all steps of the various assays without further user input. After loading the cartridge into the instrument, the user's next interaction can be to receive or view the results of the assay, either at the instrument site or remotely. The results can be as simple as an image obtained from the processed specimen, or can include automated interpretations such as microbial identification and susceptibility scores for various antimicrobial agents.

[0083] The stations may include a fluidics module for interfacing with the cartridge and manipulating the specimens and reagents therein; a magnetic pull-down station for magnetic selection of targets; a waste station for disposal of used cartridges; an imaging station for analyzing assayed specimens; incubation stations for maintaining temperature in the specimens and reagents on the cartridge as needed for the various assay steps; and a waste station for disposal of used cartridges. In preferred embodiments, the assay may use a constant temperature throughout all steps or may be modified such that the interior of the instrument may be maintained at the required temperature and performed such that the carousel may act as a storage station for incubation steps.

[0084] The instrument can read the code or tag on the cartridge or receive input from the user to determine the assay to be performed and the steps required for that assay for any given cartridge. The instrument can also use the input to record and track information associated with the specimen being tested, including patient information, for reporting results.

[0085] The instrument may include a computer including a processor and non-transitory tangible memory, and operable to schedule and control assays being performed within the instrument and to track cartridges therein. 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 assay results over the network to connected devices.

[0086] The inventive instrument may include a mechanical conveyor arm for moving cartridges between the carousel and various stations for performance of the required assay steps. In a preferred embodiment, the carousel and stations include slots sized to receive and position the cartridges within the stations. Rotation of the carousel may align the carousel slots with corresponding slots in the appropriate stations, and the mechanical conveyor arm may be operable to contact the sides 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.

[0087] The preloaded cartridges allow control of reagent volumes and dispensing at the manufacturing site, and the automated equipment controls the performance of the assay steps and their timing. Thus, the systems and methods can greatly reduce the potential for user error, allowing unskilled staff to perform a variety of assays without specialized training and obtain reliable, actionable results without the delays and costs of dedicated off-site testing.

[0088] In a preferred embodiment, the assay-specific cartridge includes a microbe-specific AST cartridge for measuring differential growth of a specimen microbe in the presence of various antimicrobial agents selected based on the specimen microbe. The robust nature of the described technology and system also allows for analysis of non-microbial targets such as cancer or other cells. For example, the present invention is useful for determining treatment efficacy, resistance monitoring (for both antimicrobials and other chemotherapeutic drugs) and treatment selection. According to the present invention, patient samples, e.g., urine, feces or blood, are analyzed directly with minimal or no specimen preparation or culture. Samples processed according to the present invention are identified and exposed to various antimicrobials or other treatment modalities (e.g., chemotherapy for cancer cells), allowing for 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. Monitoring the therapeutic effectiveness of cancer or other cells and treatments thereof using the systems and methods of the present invention allows for rapid identification of the most effective treatments, and further allows for timely intervention upon the development of therapeutic resistance.

[0089] Infection detection, target identification, and effective treatment determination are accomplished directly from patient specimens, such as saliva, urine, blood, feces, swabs, or cerebrospinal fluid, with little or no specimen preparation steps. For example, urine specimens are pipetted directly into the test device for identification (ID) and antimicrobial susceptibility testing (AST), which 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.

[0090] The systems and methods of the present invention provide images of labeled microbial target cells that allow tracking of differential growth, morphology or other observable changes to determine susceptibility and efficacy of various treatments. The specimen is directly processed and imaged to determine the presence and identity of target cells, microorganisms or pathogens present in the specimen. As described above, the processing and imaging steps occur with the specimen in a test device, such as a cartridge, requiring little to no specimen preparation outside of the test device. By discarding time-consuming specimen preparation techniques and using target-specific identifiable labels, the systems and methods of the present invention allow for the identification and enumeration of targets in a specimen in 30 minutes or less.

[0091] Specimens can also be incubated in the presence of various types and concentrations of antimicrobial agents or other treatments prior to imaging. Incubation can occur in the same test device in which the processing and imaging steps occur, or in a different test device.

[0092] The system and method of the present invention can be used to identify target cells or microorganisms and test (separately or in the same device) for therapeutic efficacy or antimicrobial susceptibility of targets in a specimen. In a preferred embodiment of the present invention, the test device divides the specimen internally into separate portions, where some of these portions can be incubated in the presence of different antimicrobial agents before imaging to determine differential growth. One or more of these portions can be directly processed and imaged to provide a baseline reference for determining growth, cytotoxicity or morphological changes in the incubated portions. By quantifying the growth of the portions incubated in different antimicrobials or other therapeutic agents, the effectiveness of each antimicrobial agent in reducing or preventing the growth of the target is determined.

[0093] The test devices and instruments described herein are not only capable of identifying and testing the effectiveness of drugs against various classes of targets (e.g., viruses, human cells, bacterial cells, or fungal cells), but also, as will become apparent through the following description, are capable of simultaneously performing such identification and therapeutic testing through the selection and manipulation of multiple different targets in a single device and multiple test devices designed for different testing applications (e.g., blood infections and respiratory infections) in a single device. Thus, robust functionality is provided by the instruments and test devices described herein.

[0094] Once a target is identified, analysis can be limited to antimicrobial agents or treatments appropriate for that target (e.g., those commonly used in treatments or those known to inhibit the growth of the identified target). As mentioned above, identification of target cells or microorganisms allows selective exposure to target-specific treatments. Thus, the initial identification step is preferably before AST or treatment efficacy testing is initiated. Identification can be performed using the same processing and imaging techniques used in the AST methods described herein, and can 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 testing can be performed on the same specimen (split into separate portions) in the same device. Target identification can also be performed using other conventional techniques, such as amplification with target-specific primers, immunoassays, mass spectrometry or oligonucleotide probe array analysis. If identification is performed separately, a dedicated differential growth analysis device can be used. A dedicated identification device compatible with the processing techniques and instruments of the present invention can dispense with the need for incubation steps or reservoirs.

[0095] Observable differential growth in the presence of various antimicrobial agents requires different amounts of time depending on the test cells or microorganisms, but is greatly reduced from the days required for standard AST 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, identification and quantification of microorganisms in a specimen can be accomplished in 30 minutes or less, thereby providing AST results within hours after introduction of the specimen into the test device. In certain embodiments, AST or therapeutic efficacy test devices are provided that contain a subset of antimicrobial or other therapeutic agents specific for various target cells or microorganisms, so that a technician can select the appropriate test device and directly introduce a patient specimen therein to perform the appropriate AST or therapeutic efficacy analysis.

[0096] The AST results obtained using the systems and methods of the present invention are then used to identify antimicrobial resistance and inform treatment decisions for patients, where the patient can be treated with the antimicrobial agent found in the AST analysis to most inhibit the growth or viability of the target.

[0097] Detectable labels may include fluorescent oligonucleotide or antibody probes, specific and non-specific ligands, lectins, dyes, and dyes that bind to targets. In certain embodiments, 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, incorporated herein by reference, may be used in the separation and imaging steps to reduce background signals and provide more accurate quantification of labeled microorganisms. Digital non-magnified imaging techniques, as described in U.S. Pat. No. 9,643,180 and U.S. Pat. No. 8,021,848, incorporated herein by reference, may be used to quantify labeled microorganisms, including single cells, for example.

[0098] An aspect of the invention provides a method for determining antimicrobial susceptibility or other response to treatment of targets in a sample. Such a method preferably includes obtaining a tissue or bodily fluid sample suspected of containing one or more types of targets. The sample is then introduced into a test device, divided into multiple portions, and incubated in the presence of multiple different antimicrobial agents, all within the test device. Also within the test device, the targets within the incubated portions are labeled and imaged to determine which antimicrobial agents inhibit the growth of the targets.

[0099] The method of the present invention may include identifying target 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 cells or microorganisms preferably includes exposing a first specimen from a patient to a magnetic tag and a fluorescent label that can bind to the 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 the detectable label, where the presence of the detectable label indicates the presence of the first target. The identification step may take less than about 30 minutes.

[0100] The specific formation of the magnetically tagged and fluorescently labeled target complex can occur 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 a specimen. There are various mechanisms by which the magnetic tag and the fluorescent label can associate with the target.

[0101] There are various ways that magnetic tags or fluorescent labels can bind non-specifically to targets. For example, binding of a binding magnetic particle (or fluorophore) to a site conserved across various categories of targets (e.g., peptidoglycan, LPS, or glucan) can be achieved by conjugating the magnetic particle (or fluorophore) to an antibody that binds to that site. Magnetic tags or fluorescent labels can also bind non-specifically due to common chemical properties. For example, positively charged magnetic particles or fluorophore labels can bind non-specifically to bacterial cells, which are generally negatively charged. Cells can be non-specifically labeled with various dyes (e.g., calcofluor) or fluorogenic dyes (propidium iodide, fluorescein diacetate).

[0102] 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.

[0103] In certain embodiments, the labeling and imaging step includes exposing the specimen portion to a target-specific binding molecule, a magnetic particle and a detectable label, where the target-specific binding molecule forms a complex comprising the target, the magnetic particle and the detectable label; applying a magnetic field to the test device to attract the complex to the detection surface; and imaging the detection surface. The detectable label can include a fluorescent marker, and the target-specific binding molecule can include an oligonucleotide probe. The exposing and imaging step can include fluorescent in situ hybridization (FISH) analysis.

[0104] The method of the present invention may include a step of determining a recommended antimicrobial or other treatment for the patient, which usually includes an antimicrobial or other treatment determined to inhibit the growth of the target. The determination of the treatment to inhibit the growth of the target may occur less than about 4 hours after the specimen is introduced into the test device. The body specimen may be a tissue specimen (e.g., a biopsy from the patient) 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.

[0105] In certain embodiments, 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 operable to receive a specimen containing a body fluid and target cells or microorganisms from a patient. 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 the portions in the test device and quantify the target complexes to determine which treatment(s) inhibit the replication of the target cells.

[0106] The system may further include a first test device operable to accept a first sample from a patient, the instrument further operable to expose the first sample from the patient to a magnetic tag and a detectable label selected to specifically form a complex between the first target, the magnetic tag and the detectable label; apply a magnetic field to the test device to attract the complex to a detection surface; and image the detection surface to detect the detectable label, where the presence of the detectable label indicates the presence of the first target.

[0107] The instrument may be operable to expose the incubated specimen portion to a magnetic tag and a detectable label selected to specifically form a complex between the target, the magnetic tag and the detectable label; apply a magnetic field to the test device to attract the complex to a detection surface; and image the detection surface.

[0108] An embodiment of the present invention provides a method for determining the therapeutic sensitivity of target cells or microorganisms in a specimen. The method may include the steps of: identifying one or more targets in a first specimen from a patient using an identification assay performed by the instrument; selecting one or more therapeutic agents based on the identified target cells or microorganisms in the first specimen; incubating the specimen from the patient separately in the instrument in the presence of each of the therapeutic agents; using the instrument to detect a number of target cells, CFU, viral load, viable target cells, or other indicators of therapeutic efficacy (or treatment effect) in each of the incubated specimens; and determining the susceptibility (or treatment effect) of each of the identified targets to each of the therapeutic agents (or treatments) based on the detected amount, morphology, or other phenotypic characteristics of the targets.

[0109] The identification assay may include contacting a first specimen with a magnetic tag and a detectable label selected to specifically form a complex of 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 presence of the complex, where the presence of the complex identifies the target present in the specimen.

[0110] Detecting the amount 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.

[0111] The multiple portions may be combined with the same antibiotic present at different concentrations, preferably corresponding to two-fold serial dilutions of the antimicrobial. In this manner, it is possible to determine the level of antibiotic that inhibits the growth of the target. The number of such portions and the concentration of the antimicrobial may be selected to obtain susceptibility / resistance results, categorical (susceptible, intermediate resistance, resistance or SIR results) or minimum inhibitory concentration (MIC) results. Appropriate concentrations of antimicrobials for specific microbial pathogen species are described by the Clinical Laboratory Standards Institute (CLSI).

[0112] In certain embodiments, the present invention provides a cartridge. The cartridge includes an incubation well; a species-specific microbial probe; and a permeabilizing agent. When a specimen containing a microorganism is delivered into the mixing well, the permeabilizing agent facilitates the entry of the probe into the microorganism while the specimen is maintained at a temperature below about 40° C. The probe may include a fluorescently labeled oligonucleotide complementary to a segment of ribosomal RNA of a specific bacterial species. The permeabilizing agent may include one or more detergents (e.g., CHAPSO, SB3-12, TRITON® X100). Preferably, the permeabilizing agent and the probe are provided in lyophilized beads that are rehydrated and dissolved by delivery of the specimen into the incubation well.

[0113] In some embodiments, the cartridge includes magnetic particles that bind to bacterial cell surfaces and a dye cushion adjacent to the transparent wall. When a magnetic field is applied across the dye cushion, the magnetic field attracts the magnetic particles through the dye cushion to the transparent wall. The dye cushion may include a solution (optionally dried or lyophilized) of density gradient medium (e.g., iodixanol or polyvinylpyrrolidone coated colloidal silica particles) that further includes a dye that absorbs light from unbound probes. Preferably, the dye cushion and the transparent wall are provided in an imaging well in fluid communication with the incubation well. The provided dye cushion may be provided in a dried or lyophilized state in the imaging well in the cartridge until it is wetted by the specimen. The magnetic particles may be linked to chemical groups that bind to bacterial cell surfaces (e.g., diethylamine ethyl starch; dextran sulfate; polyaspartic acid; polyacrylic acid; polyglutamic acid; polystyrene sulfonate; or poly-diallyldimethylamin), and the cartridge may further be preloaded with a compound that promotes the binding of the chemical groups to the bacterial cell surface. Compounds that facilitate attachment of chemical groups to cell surfaces can include cetrimide.

[0114] The cartridge may further include multiple paired imaging well / incubation well sets parallel to one another. The cartridge may include a receiving reservoir into which a user can pipette a sample into the cartridge.

[0115] In certain embodiments, the cartridge includes a slidable gate that includes a gasket with a channel therethrough. When the gate is positioned in a first position, the receiving reservoir is in fluid communication with at least the first incubation well. When the gate is in a second position, the receiving reservoir, the first incubation well, and the first imaging well are all sealed from each other. When the gate is in a third position, the first incubation well and the first imaging well are in fluid communication with each other. The cartridge may include a fitting for connecting to an external device to receive air pressure therefrom to split the specimen from the receiving reservoir into the incubation wells and subsequently pass liquid from the incubation wells into the corresponding imaging wells.

[0116] In a preferred embodiment, the probe comprises a fluorescently labeled oligonucleotide complementary to a segment of the ribosomal RNA of a particular bacterial species. Optionally, the cartridge also comprises at least one helper probe oligonucleotide that binds to the ribosomal RNA at a location within 1-30 bases of the segment. Preferably, the fluorescently labeled oligonucleotide is between 10 and 18 bases in length and comprises at least one conformationally constrained nucleic acid. In a preferred embodiment of the cartridge, the reagent composition, probe, helper probe and compound are provided as lyophilized beads that are rehydrated and dissolved upon delivery of the analyte into the cartridge; the dye cushion comprises a solution of density gradient medium further comprising a dye that absorbs light from unbound probes; the cushion is provided in a dried or lyophilized state in an imaging well in the cartridge until wetted by the analyte. [Brief description of the drawings]

[0117] [Figure 1] FIG. 1 shows a cartridge of the present disclosure.

[0118] [Diagram 2]FIG. 2 shows an instrument for use with the cartridge.

[0119] [Diagram 3] FIG. 3 shows the hardware within the device.

[0120] [Figure 4] FIG. 4 shows the transfer of the sample into the cartridge.

[0121] [Diagram 5] FIG. 5 shows the loading tray of the instrument.

[0122] [Figure 6] FIG. 6 illustrates a method for identifying microorganisms.

[0123] [Figure 7] FIG. 7 shows the steps of the microbial identification method.

[0124] [Figure 8] FIG. 8 illustrates permeabilizing agents.

[0125] [Figure 9] FIG. 9 shows species-specific probes.

[0126] [Figure 10] FIG. 10 shows the secondary structure of rRNA.

[0127] [Figure 11] FIG. 11 shows microorganism-binding magnetic particles.

[0128] [Figure 12] FIG. 12 shows cells with bound magnetic particles being separated from unbound probe.

[0129] [Figure 13] FIG. 13 illustrates the workflow for FISH at physiological temperature.

[0130] [Figure 14] Figure 14 shows the limit of detection (LoD) for E. coli ATCC 19138 is shown. The upper limit of blank (LoB) was 89 CFU per assay and the LoD was 284 CFU per assay, corresponding to a LoD of 9,467 CFU per ml of urine.

[0131] [Figure 15] Figure 15 shows the limit of detection (LoD) for P. aeruginosa ATCC 9721 is shown. The upper limit of blank (LoB) was 104 CFU per assay and the LoD was 506 CFU per assay, corresponding to a LoD of 16,867 CFU per ml of urine.

[0132] [Figure 16] Figure 16 shows the limit of detection (LoD) for K. pneumoniae ATCC 700603 is shown. The upper limit of blank (LoB) was 109 CFU per assay and the LoD was 319 CFU per assay, corresponding to a LoD of 10,633 CFU per ml of urine.

[0133] [Figure 17-1] FIG. 17 is a table of probe sequences used in the examples. [Figure 17-2] Same as above.

[0134] [Figure 18] FIG. 18 shows the percentage of input cells.

[0135] [Figure 19] FIG. 19 is a table providing the inclusion results for four additional bacteria.

[0136] [Figure 20] FIG. 20 is a table showing the probe sequences used in the examples.

[0137] [Figure 21] FIG. 21 shows the bacterial species and strains tested.

[0138] [Figure 22] FIG. 22 is a table showing the probe sequences used in the examples.

[0139] [Diagram 23] FIG. 23 shows the specific detection of E. coli and the absence of detection of the eight challenge bacteria.

[0140] [Figure 24] FIG. 24 shows the specific detection of E. coli and the absence of detection of eight additional challenge bacteria.

[0141] [Diagram 25] FIG. 25 shows a portion of the entire image acquired.

[0142] [Figure 26] FIG. 26 is a table of the probe sequences used in Example 4.

[0143] [Figure 27] Figure 27 shows BIUR0017 with nitrofurantoin.

[0144] [Figure 28] FIG. 28 shows BIUR047 with cefazolin.

[0145] [Figure 29] FIG. 29 shows BIUR057 with ciprofloxacin.

[0146] [Diagram 30] FIG. 30 shows BIUR052 with trimethoprim / sulfamethoxazole.

[0147] [Diagram 31]FIG. 31 is a table of the probe sequences used in Example 6.

[0148] [Diagram 32] FIG. 32 further shows that for bacteria tested against ceftazidime (CAZ), the presence of exclusive filamentous bacteria (compare left (normal bacteria) and right (filamentous bacteria) as easily identifiable by visual inspection) was taken as an indication of impending cell death.

[0149] [Diagram 33] FIG. 33 shows how methods can be used to obtain the MIC.

[0150] [Diagram 34] FIG. 34 shows the overall performance across all strains tested.

[0151] [Diagram 35] FIG. 35 is a table of the probe sequences used in Example 7.

[0152] [Figure 37] Figure 37 shows the average fold growth of four replicates.

[0153] [Figure 39] Figure 39 shows the average fold growth of four replicates.

[0154] [Diagram 36] FIG. 36 shows the Multipath™ UTI-AST cartridge.

[0155] [Figure 37] FIG. 37 is a table showing the antibiotic concentrations tested.

[0156] [Figure 38] FIG. 38 is a table of the oligonucleotides used in Example 8.

[0157] [Figure 39] Figure 39 shows the results for BIUR0067.

[0158] [Diagram 40] Figure 40 shows the results for BIUR0084.

[0159] [Diagram 41] FIG. 41 compares the results obtained with the new AST methods.

[0160] [Diagram 42] FIG. 42 shows the MICs obtained with the novel 4 hour method for all inoculum levels for E. coli BAA-2469 (filled circles) compared to standard broth.

[0161] [Figure 41] Figure 41 is a summary of the overall essential and categorical agreement for all organisms, antibiotics, and inoculum levels.

[0162]

[0046] FIG. 42 shows MIC results for various inoculum levels obtained using the new method described herein compared to the conventional BMD method.

[0163] [Diagram 43] FIG. 43 summarizes the MIC results obtained for various inoculum levels.

[0164] [Diagram 44] FIG. 44 is a table of the probe sequences used in Example 9. [Diagram 45] [Figure 46]

[0165] [Figure 47] Figure 47 shows data for E. coli BAA-2469.

[0166] [Figure 48]FIG. 48 shows the concordance of E. coli with various inoculum levels of standard BMD of off-target microorganisms (Micrococcus luteus, Acinetobacter baumannii, Corynebacterium minutissimum).

[0167] [Figure 49] FIG. 49 shows the concordance of E. coli with various inoculum levels of the standard BMD of an off-target microorganism (K. pneumoniae).

[0168] [Figure 50] FIG. 50 is a table of the probe sequences used in Example 10.

[0169] [Figure 51] FIG. 51 shows the MICs for susceptible E. coli strains.

[0170] [Figure 52] FIG. 52 shows similar results for the lactam antibiotic meropenem.

[0171] [Figure 51] Figure 51 is a comparison of the new rapid AST method and the BMD method for determining the MIC of imipenem for E. coli in the presence of carbapenem-hydrolyzing B-lactamase-resistant strains of K. pneumoniae.

[0172] [Figure 52] Figure 52 shows that E. coli MICs are consistent with the above method for various inoculations of carbapenem-hydrolyzing B-lactamase-resistant strains of K. pneumoniae, while standard BMDs are not.

[0173] [Diagram 53] FIG. 53 is a table of the probe sequences used in Example 11.

[0174] [Figure 54] FIG. 54 shows the MIC.

[0175] [Figure 55] Figure 55 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.

[0176] [Figure 56] FIG. 56 shows the determined MICs.

[0177] [Figure 57] FIG. 57 shows the results obtained from the study.

[0178] [Figure 58] FIG. 58 is a table of the probe sequences used in Example 12.

[0179] [Figure 59] FIG. 59 shows S. aureus cells (left panel) and TSB medium only (right panel).

[0180] [Figure 60] Figure 60 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).

[0181] [Figure 61] FIG. 61 shows the categorical determinations of susceptibility and resistance.

[0182] [Figure 61] Figure 61 shows ciprofloxacin-susceptible and -resistant strains used in the Examples.

[0183] [Figure 62]FIG. 62 is the first half of a table of probe sequences used in Example 13.

[0184] [Figure 63] FIG. 63 is the second half of a table of probe sequences used in Example 13.

[0185] [Figure 64] Figure 64 shows the essential agreement for polymicrobial infections with two target organisms. As can be seen below, the AST method gives an essential agreement of 100% with the standard BMD.

[0186] [Figure 65] Figure 65 shows the category agreement for polymicrobial infections with two target organisms. As can be seen below, the AST method provides 100% category agreement with standard BMD.

[0187] [Figure 66] FIG. 66 shows that the target pathogens are detected only in wells containing their species-specific DNA oligonucleotide FISH probes.

[0188] [Figure 67] FIG. 67 shows that the target pathogens are detected only in wells containing their species-specific DNA oligonucleotide FISH probes.

[0189] [Figure 68] FIG. 68 shows that the target pathogens are detected only in wells containing their species-specific DNA oligonucleotide FISH probes.

[0190] [Figure 69] FIG. 69 is the table "Table A of Example 14," which shows that the target pathogens were detected while other non-target pathogens were not detected.

[0191] [Figure 70] FIG. 70 is a table, “Table B of Example 14,” showing the probe sequences used in Example 14. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0192] The present invention provides a cartridge that works in conjunction with an automated instrument to automatically perform antimicrobial susceptibility testing (AST) analysis of target microorganisms in a specimen, requiring little or no specimen preparation by the user. Target cells, e.g., cancer cells, can also be identified and the therapeutic efficacy of various treatments can be evaluated using the systems and methods of the present invention. Cells or microorganisms in a specimen can be analyzed for differential growth in the presence of various antimicrobial agents, or the effect of therapeutic agents on cell viability, morphology, viral load, cell functionality, or other measures of therapeutic efficacy can be determined using the systems and methods described herein. Specimen manipulation, incubation, processing, and analysis steps are performed within a single test device in less than about 30 minutes for target cell identification and less than about 4.5 hours for AST analysis for urine analysis for common causes of urinary tract infection. 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.

[0193] The present invention allows for testing without the need for cell purification steps by the user. By reducing extraneous steps and using imaging methods and image analysis capable of quantifying single cells or colony forming units (CFUs) in certain cases, actionable results directly from patient specimens for target cell or microbial identification and AST analysis can be obtained in just a few hours, compared to multiple days using conventional techniques. The test device can be automatically manipulated by the instrument to perform each of the steps of splitting 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 tested antimicrobial agents.

[0194] In a preferred embodiment of the present invention, the cartridge is used for antimicrobial susceptibility testing, and the specimen is divided into separate portions containing a nutrient growth medium that promotes the replication or growth of microbiological cells. One or more of these portions can be used as a reference or baseline portion that is directly processed and analyzed before incubation at a temperature that promotes growth to determine the number and quality of pathogen cells. One or more of these portions can be incubated at a temperature that promotes the growth of pathogen cells to determine whether the pathogen cells are viable. Additionally, each of the other portions contains a specific concentration of one or more antimicrobial agents and is incubated to determine the effect of the antimicrobial agents on pathogen cell replication.

[0195] The present invention can then be used to analyze the number and quality of pathogen cells in the incubated portion and compare these results with the number and quality of pathogen cells in a non-incubated reference portion.If the ability of a pathogen cell to replicate normally in a portion containing a particular antimicrobial agent is significantly impaired, the pathogen will be scored by the analysis software as being sensitive to the concentration of the antimicrobial agent in that portion.Alternatively, if normal growth is not significantly impaired in that portion, the pathogen will be scored by the analysis software as being sensitive to the concentration of the antimicrobial agent in that portion.

[0196] The present invention may include evaluation criteria, for example, cell replication is evaluated for determination of antimicrobial susceptibility or resistance of a pathogen or microbial target in a portion containing an antimicrobial agent(s). These criteria may be determined for a particular combination of parameters, including, but not limited to, target microbial species, specimen type, antimicrobial agent, growth medium composition, temperature, and incubation time.

[0197] The evaluation criteria are preferably empirically determined by correlation with accepted reference methods for antimicrobial susceptibility testing. One standard reference method is broth microdilution (BMD), which is understood by those skilled in the art. Broth microdilution is a method in which the antimicrobial susceptibility of microbial strains to antimicrobial agents is evaluated under standard conditions. Purified cells of the target microorganism are added at a defined concentration to a series of portions or aliquots of a defined nutrient growth medium containing two-fold serial dilutions of various antimicrobial agents. The antimicrobial susceptibility of the microbial strain is determined after visually evaluating the turbidity of the various portions after a defined period of growth incubation. The lowest concentration of the antimicrobial agent at which turbidity is visually absent or significantly reduced compared to the portion without the antimicrobial agent is called the minimum inhibitory concentration (MIC). Organizations that determine standards for antimicrobial susceptibility testing (e.g., CLSI or EUCAST) correlate MIC values ​​for a combination of a specific microbial species and a specific antimicrobial agent with the effectiveness of a specific therapeutic dose of the antimicrobial agent in clinical practice. MIC values ​​are commonly binned into categorical ranges: susceptible, intermediate resistance, and resistance. These are called SIRs or categorical antimicrobial susceptibility test results. In this way, the MIC for a particular strain of a particular species against a particular antimicrobial agent can be reported as a SIR or categorical result. Other standard methods used to determine include -Bauer or disk diffusion and agar dilution. These methods are described in CLSI and EUCAST documents and are known to those skilled in the art.

[0198] The evaluation criteria for determining antimicrobial susceptibility test results using the present invention are empirically determined by using the present invention to evaluate the degree and quality of cell replication of various strains of a particular species at various concentrations of a particular antimicrobial agent, similar to broth microdilution. The criteria for assigning antimicrobial susceptibility test results (generally MIC or SIR categorical results) to strains of a particular species for a particular antimicrobial agent are selected so that the results for various strains are consistently correlated with the results of the reference broth microdilution method using the present invention. For example, a criterion that can be used by the present system and method for determining antimicrobial susceptibility test results is the evaluation of the growth fold (the fold increase in the number of target cells) of a target microorganism of a particular species over a certain period of incubation in a nutrient growth medium at a certain temperature in the presence of various concentrations of a particular antimicrobial agent. In this case, the empirical study to determine the effective criteria for use with the present system and method for evaluating antimicrobial susceptibility evaluates the growth fold of various strains of a species at various concentrations of an antimicrobial agent. A threshold for growth fold can be selected such that the growth fold measured for a strain using the present invention correlates with the broth microdilution results for the same strain grown in the presence of the same antimicrobial agent. The threshold is selected empirically using various strains of the target species such that if the growth fold of a strain exceeds the threshold, the strain is categorized by the present invention as having significant growth in the presence of that concentration of antimicrobial agent. If the growth fold of a strain is below the growth fold threshold, the strain is categorized by the present invention as not having significant growth. Thus, in this example, the threshold for growth fold is selected such that both the reference broth microdilution method and the method of the present invention return the same results as to whether various strains are determined to have significant growth or not at various antimicrobial agent concentrations.

[0199] Other evaluation criteria can also be used by the present invention to determine antimicrobial susceptibility test results. For example, the present invention can include evaluation of morphological features that reflect the disruption of normal cell replication caused by incubation in an antimicrobial agent. As another example, the degree of growth inhibition in the portion containing the antimicrobial agent compared to the portion not containing the antibiotic after the incubation step can be evaluated. Multiple evaluation criteria can also be used simultaneously to determine whether a particular concentration of the antimicrobial agent causes significant disruption to normal cell replication of the target cells. Thus, the present invention can be used to detect infections, identify infectious pathogens, and determine which antimicrobial agents are effective for treatment directly from patient specimens.

[0200] The patient specimen, e.g., urine, feces, respiratory, wound, cerebrospinal fluid or blood, is preferably transferred directly into the analysis cartridge for microbial analysis without any specimen preparation by the user. Thus, there is preferably no need for the user to perform colony purification, nucleic acid purification, or other time- or labor-intensive specimen preparation protocols to isolate a large number of pure pathogen cells. The specimen is preferably loaded directly into the test cartridge without prior enrichment or purification, e.g., to remove biological debris. The cartridge contains the reagents for microbial quantification and identification and antimicrobial susceptibility testing of the present disclosure. All steps, such as species-specific labeling and imaging of the microorganisms in the specimen, are performed on the cartridge on which the specimen is directly loaded. The cartridge contains target cell-specific labels, e.g., fluorescent probes, that are used to identify the microorganisms in the specimen by the systems and methods of the present invention. To identify the microorganisms, the instrument preferably includes an imaging subsystem for imaging the labeled microorganisms in the cartridge.

[0201] Rapidly detecting infections, identifying pathogens, and determining antimicrobial susceptibility using the systems and methods of the present invention offers the potential for actionable results that guide effective treatment of patient infections much sooner than traditional methods that require lengthy culture steps. The systems and methods of the present invention can provide clinicians with the ability to detect infections and identify infectious pathogens in approximately 30 minutes, and determine antimicrobial susceptibility test results in a few hours, by simply transferring the patient specimen directly into the analysis cartridge and loading the cartridge into the instrument.

[0202] In some embodiments, the present invention includes microbial detection and identification of effective treatments directly from patient specimens. In certain aspects, the present invention provides for identification of microorganisms. Microorganism identification is achieved directly from patient specimens, such as whole blood, plasma, serum, urine, sputum, saliva, feces, cerebrospinal fluid, amniotic fluid, peritoneal fluid, pus, lymph, vaginal secretions, nasal secretions, vomit, sweat, and tissue, without a specimen preparation step. For example, the method may include transferring a urine specimen directly into an analytical cartridge device for microbial identification. In some embodiments, the method includes transferring a patient specimen directly into a sample well of an analytical cartridge, and operating the cartridge to label the microorganisms in a species-specific manner, image the labeled microorganisms, and identify the labeled microorganisms.

[0203] In various embodiments of the present invention, microorganisms in a specimen are tagged with magnetic particles and labeled with species-specific detectable labels. In some embodiments, the detectable labels may include species-specific fluorescent nucleic acid probes. Other detectable labels include target-specific fluorescent antibodies or aptamers, members of receptor-ligand pairs, lectins, or dyes. The target-specific nucleic acid probes may be selected to be complementary only to the nucleic acid sequence of the target microorganism. The magnetic particles used in various embodiments may bind specifically or non-specifically to the target microorganism. In some embodiments, multiple species may be detected.

[0204] In some embodiments, operating the cartridge may include loading the cartridge into an instrument to detect infection, identify and quantify pathogens in a specimen, and determine antimicrobial susceptibility within the cartridge device of the invention. The present invention provides analytical cartridges and instruments capable of directly receiving and processing patient specimens to identify microorganisms and / or determine treatment susceptibility and efficacy, all within the cartridge.

[0205] In some preferred embodiments, separate cartridges are deployed for different sequential diagnostic functions. For example, in a preferred embodiment, the cartridge detects infection, identifies the pathogen, and quantifies the pathogen in the specimen. Once an infection is detected and the pathogen is identified, another portion of the patient specimen is tested on a cartridge containing antimicrobial agents that are candidates for treating the identified pathogen. Using the systems and methods of the present invention for antimicrobial susceptibility testing, the cartridge can be used to determine which antimicrobial agents may be effective for treating a particular strain of a pathogen causing an infection in a patient.

[0206] The cartridges of the present invention can also be used for the sensitive detection of biologically important molecules, such as toxins and biomarkers.

[0207] The systems and methods of the present invention include instruments or analyzers that can be used to interact with the analytical cartridge to perform the methods of the present invention. The instruments can include multiple subsystems for performing the methods of the present invention. The analytical cartridge can be loaded into an instrument that has multiple subsystems for processing the specimen in the cartridge. In a preferred embodiment, one of the multiple subsystems can be an imaging subsystem for imaging the labeled microorganisms in the cartridge. The instrument subsystems can also include a pneumatic subsystem, a magnetic subsystem, and a waste subsystem. The instruments can also include a carousel, a pusher mechanism, and a task scheduler to move and manipulate the cartridge within the instrument. The instruments are capable of performing all of the processing steps at a constant temperature.

[0208] The systems and methods of the present invention, including instruments and devices, can perform a wide range of diagnostic functions, including detecting infection; detecting, identifying and quantifying all types of pathogen cells; determining antimicrobial susceptibility; detecting and quantifying biomarkers, including toxins, viruses, and host-response factors; and detecting and quantifying diagnostically informative human or host cells. The systems and methods of the present invention are capable of performing such diagnostic functions, alone or in combination, simultaneously on a single specimen in a single device (e.g., analytical cartridge). The systems and methods of the present invention include the capability of random access processing, such that multiple such devices performing different types of diagnostic tests (e.g., for urinary tract infections, blood infections, and respiratory infections) can be processed simultaneously on a single instrument. Thus, the instruments and cartridges described herein provide such functionality and can be manipulated accordingly to process specimens within the device.

[0209] The AST cartridge may include a series of interconnected compartments preloaded with the necessary reagents for the assay. The user need only add the specimen from the patient directly to the cartridge based on the desired assay and specimen type, and then load the cartridge into the instrument for automatic processing. The instrument can identify the type of assay to be performed through user input or through reading the cartridge itself (e.g., scanning a code thereon). The instruments described herein may include various stations for performing the various steps that may be required for different assays, and may include a carousel or other mechanism for storing multiple in-process cartridges and moving them between stations as needed for the performance of the assay steps.

[0210] The cartridge may include various valves and channels to connect different compartments therein as needed to perform the steps of the desired assay. For example, a cartridge for an AST assay may include a series of growth reservoirs preloaded with growth medium and different antimicrobials to be analyzed. The cartridge may also include compartments for processing the post-growth specimen and labeling the targets therein, as well as imaging wells that provide windows for imaging the labeled targets. The instrument may include a fluidics module that interfaces with the cartridge to allow external manipulation of valves and pressures in the cartridge to connect different compartments and move specimen volumes between them as needed for various assays.

[0211] FIG. 1 shows a cartridge 101 for use in various systems and methods of the invention. The test cartridge 101 includes a specimen well 103 for receiving a specimen, a dividing well 105, a reagent well 109, an imaging well 111, and a channel 113 for moving the specimen between the wells, as well as a sliding valve 107 for controlling the movement. The cartridge 101 may contain reagents for a specific assay. For example, in the reagent well 109, the cartridge may contain target-specific probes, permeabilization reagents or other materials, which may be provided contained in beads 141 (e.g., lyophilized beads).

[0212] As shown, the cartridge may include multiple paired imaging / incubation well sets parallel to one another. Here, cartridge 101 is shown to include eight parallel "channels," each including a separation well 105, a reagent well 109, and an imaging well 111. An embodiment of the cartridge may include two groups of eight channels (with an additional eight channels behind the eight visible channels), resulting in 16 channels overall in one cartridge. The cartridge may be described according to its dimensions, such as height h, length l, and width w (where w is measured perpendicular to h and l). Height h may be between about 3 cm and about 10 cm. Length l may be between about 5 cm and about 12 cm. Width w may be between about 0.5 cm and about 3 cm. For example, in one embodiment, h is about 6 cm, l is about 8 cm, and w is about 2 cm.

[0213] The cartridge 101 preferably includes a specimen well 103 through which a user can pipette a specimen into the cartridge. In one particular embodiment, the cartridge 101 includes a sliding valve 107 that includes a gasket having a channel therethrough. When the sliding valve 107 is positioned in a first position, the specimen well 103 is in fluid communication with at least the first divided well 105. When the sliding valve 107 is in a second position, the specimen well 103, the first divided well 105, and the first reagent well 109 are all sealed from one another. When the sliding valve 107 is in a third position, the first divided well 105 and the first reagent well 109 are in fluid communication with one another.

[0214] The cartridge 101 may include fittings for connection to external equipment to receive air pressure therefrom for dividing (hence "dividing") the specimen from the specimen well 103 into the dividing well 105 and subsequently passing the liquid from the dividing well 105 into the corresponding reagent well 109. The imaging well 111 preferably includes a dye cushion 115 and a transparent window (e.g., in the bottom of the cartridge 101).

[0215] The dye cushion 115 is a material at the bottom of the imaging well 111. When a portion of the analyte is transferred into the imaging well 111, it forms a liquid analyte layer, and the dye cushion 115 underlies the liquid analyte layer. The dye cushion is preferably a material that resists migration of particles (e.g., density media, gels, etc.) and inhibits the passage of light (e.g., via containing pigments or dyes). Due to the dye cushion 115, the probes are attracted to the detection zone only if they bind to targets that are also bound to magnetic beads. The dye cushion 115 excludes unbound material from the window that provides the detection zone (because it exists as a cushion layer underlying the liquid analyte layer) and inhibits light from unbound signaling moieties from reaching the detection zone (because the dye cushion layer contains dyes and is not transparent).

[0216] The cartridge 101 can interface with the fluidics module of the instrument via a pneumatic port 117. The sliding valve 107 can control the movement of fluid between the divided wells 105 and the reagent wells 109 by opening and closing the connection. The sliding valve 107 can allow one or more portions of the specimen to bypass the divided wells 105 and proceed directly to the reagent wells 109 and imaging wells 111, for example, to provide a zero growth reference for AST analysis, or in applications focused on identifying the presence of target cells or microorganisms but unrelated to AST analysis. The sliding valve 107 can be manipulated by the fluidics module of the instrument 201. In a preferred embodiment, the cartridge 101 includes a diluent reservoir 121 and a button 127. Pressing the button (e.g., manually by an operator or mechanically within an instrument of the present disclosure) dispenses a diluent (e.g., saline) preloaded in the diluent reservoir 121 into the divided wells 105. In other embodiments of the invention, valve 107 may also be configured to allow one or more portions to bypass distribution well 105 and proceed directly to one or more reagent wells 109 and imaging well 111 to provide a zero growth reference or baseline for AST analysis. Distribution well 105 may be pre-filled with growth medium and / or one or more antimicrobial agents. Valve 107 may hold specimen portions in distribution well 105 to be incubated in the presence of various antimicrobial agents for any period of time.

[0217] 2 shows an instrument 201 (e.g., an analyzer) for performing microbial identification and antibiotic susceptibility testing (AST) using cartridge 101. The instrument 201 can be used to interact with cartridge 101 to perform target cell identification and AST analysis of a specimen. A lift-up opening loading door 202 can be included to provide access to a loading tray.

[0218] The instrument 201 includes at least one user interface 203 (e.g., a touch screen) for displaying prompts, results, reports, and receiving commands. The instrument 601 may include different functional areas and subsystems. The compartments may include a carousel 205 for transporting and incubating analytical cartridges, an upper compartment 207 housing processing and incubation equipment, and a lower compartment 209 housing electronics, imaging, and pneumatic equipment. The instrument 201 and methods of the present disclosure may be used to identify and quantify a variety of target cells or microorganisms, including viruses, bacteria, fungi, parasites, human cells, animal cells, plant cells. By matching the growth medium and antimicrobial agents to the target cells or microorganisms, AST analysis may be performed on a variety of target cells.

[0219] 3 is an exemplary top view of the instrument 201. The instrument 201 can include an input mechanism 303, e.g., a loading tray, for receiving and sorting multiple analytical cartridges 101.

[0220] The instrument 201 may include a carousel 205 and a mechanical conveyor arm 307 for receiving, moving, and manipulating the cartridges 101 within the instrument 201. The instrument 201 may also include a task scheduler. The instrument 201 is preferably computer controlled to automate the manipulation of the analytical cartridges, the performance of the microbial identification and AST analysis, and the generation of results. The instrument 201 may include multiple subsystems for carrying out the methods of the present invention.

[0221] The subsystems of the instrument 201 may include a pneumatic subsystem 309, a magnetic subsystem 311, an imaging subsystem 313, and a waste subsystem 315. The magnetic subsystem 311 may include, for example, a permanent magnet or an electromagnet that provides a magnetic field to deposit the magnetic particle-target complexes on the detection surface of the analytical cartridge for imaging. The imaging subsystem 313 may be, for example, those described in U.S. Pat. Nos. 9,643,180 and 8,021,848 as discussed above, as well as a stage for manipulating the detection surface of the analytical cartridge 101 relative to the imaging module of the instrument 201. The imaging subsystem 313 may be operatively associated with a computer to provide image processing, analysis, and display performance. The pneumatic subsystem 309 may be operable to drive the movement of specimens and reagents within the analytical cartridge 101, for example, via manipulation of the sliding valve 107 (e.g., plunger and actuator) using functionality provided by air pressure or vacuum. In some embodiments, hydraulic or mechanical means may also be incorporated into the pneumatic subsystem 309 to effect analyte movement. The waste subsystem 315 may include a receptacle (e.g., a removable bin) for disposal of the cartridge 101 after use.

[0222] The instrument 201 may also include one or more incubation areas for holding (or storing) analytical cartridges during growth incubation and / or assay incubation. The incubation areas may include heating and / or cooling elements and thermostats to control the elements to maintain the incubation area at a desired temperature (e.g., 35° C.) for growth of target cells or microorganisms or to perform assay incubation.

[0223] In some embodiments, the mechanical conveyor arm 307 may be operable to manipulate the analytical cartridge 101 among various subsystems within the instrument 201. In some embodiments of the invention, the mechanical conveyor arm 307 moves each of the analytical cartridges 101 between the carousel 205 and various subsystems of the instrument. The mechanical conveyor arm 307 applies a pushing force to move the analytical cartridge 101 toward and away from the carousel 205. The carousel 205 may rotate to position the analytical cartridge 101 adjacent to another subsystem, and then the mechanical conveyor arm 307 may apply a force to slide the analytical cartridge 101 onto the subsystem. The analytical cartridge 101 is preferably never gripped, for example, by a squeezing or compressing force within the instrument 201. Sliding or pushing the analytical cartridge 101 within the instrument 201 reduces exposure to debris. The various stations or subsystems within the instrument, as well as the carousel 205, may include slots 317 sized to receive the cartridge 101 and guide the cartridge 101 as it is slid between the carousel 205 and the various stations. Rotation of the carousel 205 serves to align the slots 317 thereon with corresponding slots on the stations such that a guide or track is formed along which the cartridge 101 may be slid by the mechanical conveyor arm 307.

[0224] In some embodiments, the instrument 201 includes a task scheduler for managing the analytical cartridge 101. The task scheduler is operable to control the movement, such as the transportation and movement, of each of the analytical cartridges 101 among the multiple subsystems. In some embodiments, the time each analytical cartridge 101 spends in the subsystems may also be managed by the task scheduler. The task scheduler may set aside time on the various subsystems as necessary for each analysis of the analytical cartridge 101. In some embodiments of the present invention, the task scheduler may manage the movement of the analytical cartridge 101 (i.e., the steps / parameters of the analysis to be performed) by identifying the contents of the cartridge. The scheduler may include software stored on a tangible non-transitory memory and operated by a processor. The processor may be in and / or communicate with the instrument 201 and various motors and subsystems or stations thereon, for example, to operate the carousel and mechanical conveyor arm mechanisms, as well as to control the imaging devices and record images in memory as they are received from the imaging subsystems or stations. The processor and memory may comprise a computer, which may also include input / output devices 203, such as a monitor, keyboard, mouse, or touch screen.

[0225] In some embodiments, the instrument 201 may also include a reader operable to analyze an identifier (e.g., a barcode) located on the analytical cartridge 101. The contents and required processing of the analytical cartridge 101 may be associated with the identifier on the analytical cartridge 101. Each of the analytical cartridges 101 may include an identifier readable by the instrument 201. The instrument 201 may read the identifier via the reader and associate the identifier with a particular set of instructions for executing the task scheduler.

[0226] Upon reading the identifier, the computer processor can access the assay associated with that identifier (e.g., AST analysis for E. coli). The processor can then determine a schedule for performing the required steps of the assay, and upon initiation, can determine when each station or subsystem is required and how long it will take to complete the assay. 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., incubations) may be flexible and the schedule may provide for different lengths that can be altered 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 assay 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 high flow rate station, e.g., a fluidics module, may warrant including two or more, depending on the desired capabilities of the instrument.

[0227] The instrument 201 includes a user interface 203 for receiving user input and displaying results, status, and other information. The instrument 201 is enclosed to maintain a desired incubation or reaction temperature within the instrument 201. The instrument has an access door that is open to allow access to a loading tray 303 where a user can load a cartridge for analysis. The instrument 201 can read the identifiers on the cartridges in the loading tray 303 before opening the interior door to begin processing and bringing the cartridge into the carousel. Thus, if there are any errors or scheduling conflicts, they can be addressed before the cartridge is installed. The loading tray 303 positions the cartridge in a set location relative to the instrument to allow the instrument to scan a known location for the identifier and for the mechanical conveyor arm mechanism that engages the cartridge and brings it into the carousel. The cartridge and loading tray may include an asymmetric footprint so that the cartridge can only be inserted into the tray in one orientation to avoid jams or errors, such as the identifier pointing to a location that is out of line with the instrument's scanner.

[0228] The instrument 201 and carousel 205 operate to perform tests using the cartridge 101. Using the instrument 201, the cartridge 101 is useful for performing rapid antibiotic susceptibility testing (AST) from polymicrobial specimens based on differential growth and species-specific detection. Differential growth useful for determining AST can be observed after incubation of many target cells and microorganisms in the divided wells 105 for only a few hours. In various embodiments, specimen portions can be incubated in the divided wells 105 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. Growth media and / or antimicrobial agents can be selected based on the target cells or microorganisms to be analyzed in the specimen and included in the cartridge 101. For example, a growth medium known to support growth of the identified target cells or microorganisms and a therapeutic or antimicrobial agent commonly used to treat the identified target can be selected. In various embodiments, the cartridge 101 may be pre-loaded with growth media and therapeutic agents for a particular target such that a user, having identified a target in a specimen (e.g., having determined the source of an infection in a patient or a particular cancer type), can select the appropriate pre-packaged test device for AST analysis of the target microorganism or therapeutic efficacy analysis of target cells.

[0229] After a suitable incubation period, the sliding valve 107 can be manipulated to move the incubated specimen portion from the partition well 105 to the reagent well 109. The reagent well 109 can be pre-filled with processing reagents to label target cells or microorganisms for imaging. The processing reagents can be lyophilized for storage and activated upon contact with the fluid specimen portion and growth medium. For example, the reagent well 109 can contain species-specific detectable probes and microorganism-binding magnetic particles. As the specimen portion passes through the reagent well 109, the microorganism-binding magnetic particles bind to all microorganisms present, while the species-specific detectable probes bind to organisms of interest for detection (e.g., by microscopy).

[0230] For some applications, magnetic particles or other detectable labels, such as avidin-coated magnetic particles and SYBR-green dye, may be non-specific.For example, biotin-labeled target-specific antibodies (i.e., target-specific binding molecules) can be used to target specific cells or microorganisms, which are then 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.

[0231] The instrument 201 may use a magnetic field to pull the complexes to a detection surface on the bottom of the imaging well 111. The imaging well 111 may include a dye cushion that forms a high density opaque aqueous layer that underlies the upper assay layer in the imaging well described herein. The complexed magnetic particles are forced through the lower dye cushion layer and deposited on the detection surface of the imaging well 111. The detection surface may be crystal clear to allow optical detection of the labeled target cells or microorganisms. After processing and magnetic field pull down, the cartridge 101 may be placed on an imaging stage for imaging. Target-specific fluorescent oligonucleotide probes may be used to fluorescently label target cells such that imaging of the fluorescent label provides quantification of the target cells (e.g., cell counting for target bacteria in a specimen).

[0232] The cartridge 101 may have any number of growth or separation wells and corresponding processing and imaging wells, but preferably has at least three wells for a zero growth reference sample and differential growth analysis of at least two separate therapeutic agents. In some embodiments, the test device includes at least eight or even sixteen wells.

[0233] By counting the number of species-specifically labeled cells present in each specimen portion after differential growth in the presence of various antimicrobial agents, the effect of each therapeutic agent on a particular species can be determined. By determining which antimicrobial agent best inhibited growth, an effective therapy for treating the patient's infection can be determined.

[0234] Antimicrobial selection can be random when the bacteria or target cells in the specimen are unknown and a large number of wells and a large amount of specimen are available to allow for extensive random testing. Preferably, the bacteria or other target cells have already been identified and various antimicrobials, such as antimicrobials commonly used to treat the identified bacteria, have been selected accordingly. Each split well can contain a single antimicrobial agent or a unique combination of antimicrobial agents to evaluate the combined effect on target cell growth. Different concentrations of different antimicrobial agents can be combined with specimen portions or aliquots in different split wells, and some split wells can be used to replicate treatment with the same antimicrobial agent to enhance results. Specimen portions or aliquots in some split wells may not be combined with antimicrobial agents to quantify uninhibited growth and / or detect growth inhibition due to either non-antimicrobial inference or reagent instability.

[0235] 4 shows the transfer of a portion of a specimen into the cartridge 101. First, the loading tray 303 may be transported away from the instrument 201 and set on the benchtop. To use the cartridge 101, a specimen is placed into the specimen well 103 of the cartridge 101. This may be performed by pipetting from a specimen collection device, e.g., a specimen collection container, swab, etc. The cartridge 101 may then be placed into the loading tray 303.

[0236] 5 shows a user loading the loading tray 303 into the instrument 201 for use with the cartridge 101. Preferably, the cartridge 101 is loaded into the instrument 201 for analyzing specimens. After the cartridge 101 is placed in the instrument, air pressure or a similar force can be used to split the specimens from the specimen wells 103 into the split wells 105 via the channel 213.

[0237] The specimen may be mixed with a volume of growth medium within the cartridge 101 at any point between introduction of the specimen and incubation. The incubation 115 step may last less than about 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, or 24 hours or more to allow for measurable differential growth of target cells or microorganisms in the specimen 105, but is preferably about 4 hours.

[0238] After incubation 115, the incubated portions 117 may be exposed to, for example, magnetic tags and detectable labels that may form complexes 119 with specific target cells in the specimen portions for imaging 123. The labeled target 121 complexes may then be imaged 123 in the portions, and the images 125 may be analyzed (manually or by a computing device) to quantify the amount of labeled target 121 present in each portion.

[0239] By comparing the amount of target cells or microorganisms in the portions incubated in the presence of various antimicrobial agents, it can be determined which of those agents inhibited growth. The comparison preferably involves the amount of target cells from a "time zero" growth reference portion that was aliquoted, processed, and imaged from the original specimen without incubation.

[0240] Target microorganisms and cells contemplated for testing using the systems and methods of the present invention include viruses, bacterial or fungal cells, or human cells, such as cancer cells or beta cells. In the case of bacterial targets, for example, specimens can be incubated in the presence of various antibacterial agents, and the effects of these agents on target bacterial growth in the specimen can be analyzed to determine efficacy. The systems and methods described herein can be equally applied to measure the cytotoxic effects of chemotherapeutic agents on cancer cells, or to measure the effectiveness of various antiviral agents on viral load in a specimen. The only variations that may be required across different target cells and microorganisms are the target-specific binding molecules (e.g., bacterial cell surface-specific antibodies, virus-specific oligonucleotide probes, or cell-specific dyes), the treatment being tested (e.g., antiviral drugs, antibacterial agents, or cancer treatments), the incubation time, and in some cases, the characteristics being analyzed in the image analysis (e.g., CFU quantification, viral load, or cell number, morphology, or function).

[0241] The specimen may be inserted directly into the receiving well 103. Pneumatic port 117 may be opened to apply pressure or vacuum to dispense the specimen into the wells in the cartridge 101. The specimen may be first dispensed from the specimen well 103 into the dividing well 105, held there by the sliding valve 107 during incubation, and released into the reagent well 109 for labeling and then into the imaging well 111 for imaging. The instrument's fluidics module may interface with the cartridge to control valve movement in coordination with specimen and reagent distribution according to the requirements of a particular assay.

[0242] FIG. 6 illustrates an exemplary method 601 of the present invention. The method 601 preferably includes a step of obtaining a specimen from a patient. The specimen may include a bodily specimen from a patient, such as blood or a portion thereof (e.g., plasma or serum), urine, sputum, cerebrospinal fluid, feces, wound, or any other specimen obtained from the human body. In certain embodiments, non-medical specimens may be tested, including, but not limited to, veterinary, environmental, agricultural, and food specimens. The specimen may be delivered 607 or introduced directly into a collection tube, well, or reservoir of the cartridge without the need for prior target cell purification. For example, a direct urine specimen from a patient may be pipetted into the analytical cartridge without the need for colony purification, or specimen preparation including prior mixing with reagents, target purification, treatment to remove specimen components, centrifugation, biochemical enrichment, or other treatments. Once obtained, the specimen is introduced 607 into the analytical cartridge (e.g., device). The specimen may include biological debris and other materials. The method 601 includes incubating 613 the specimen with a label that binds only one species of microorganism, i.e., a target species-specific label. Optionally, a permeabilizing agent can be introduced to permeabilize 625 the cells in the specimen. The method 601 further includes separating 629 the cells in the specimen from unbound label, and detecting 635 the bound label in the cells to indicate the presence of that species in the specimen. Note that in certain embodiments, the label is species-specific and the cells are generally separated from unbound probe.

[0243] The present disclosure provides a cartridge preloaded with reagents for performing fluorescent in situ hybridization (FISH) that can be performed at a constant physiological temperature. The cartridge of the present disclosure can be used to probe and detect the genetic content of cells and organisms in a specimen without the need to subject the specimen to extreme heat that may otherwise lyse the cells of interest, denature proteins, unduly affect the relative differential growth rates of different cells or organisms, or otherwise have deleterious effects on the chemical components and process steps of the specimen analysis protocol. One important property of being able to perform FISH at a constant physiological temperature is that a specimen can be fluorescently probed and imaged in one cartridge while other cartridges that are simultaneously used to perform other biological assays that are optimal at physiological temperatures remain loaded on the instrument. The cartridge of the present disclosure includes all the hardware, tooling, and reagents for performing microbial identification or antibiotic susceptibility testing using FISH at physiological temperatures. If some of the test steps include incubation to promote growth of the organism of interest, the ability to simultaneously perform fluorescent probe hybridization in other wells of the same cartridge without heating significantly above physiological temperature allows multiple steps to proceed all at their own time and pace, including chronologically overlapping or even simultaneous. Furthermore, if the fluorescent probe hybridization is performed in wells within a cartridge that is loaded into and operated on an analytical instrument, the instrument can multiplex cartridges, routing and scheduling multiple different cartridges to different test steps while maintaining a constant temperature within the instrument.

[0244] A preferred embodiment of the present disclosure provides a cartridge loaded with reagents for performing a fluorescent probe hybridization protocol that operates at physiological temperatures. Generally, physiological temperature refers to the body temperature of an organism, such as an animal. The process steps, molecular species, and chemical reagents disclosed herein are useful for hybridizing fluorescent probes to nucleic acids in cells and imaging those probes at physiological temperatures without lysing the cells. There is flexibility regarding the temperature at which the specimen is exposed and the steps are performed. The methods of the present disclosure may be usefully performed at temperatures that fluctuate but do not exceed 45°C, and may even operate at temperatures that do not exceed 40°C. The methods and compositions of the present disclosure are useful and functional when used at temperatures within the range of, for example, 36-39°C. In fact, the methods of the present disclosure may be performed on an instrument that maintains a temperature essentially at or about human body temperature, i.e., about 37°C for healthy humans, 38°C for humans with fever, or 36°C for some nocturnal human temperature fluctuation patterns. By "about" we mean within a decimal point, etc. That is, 36.3 is considered to be about 36.5, and 37.7 is considered to be about 37.5. It is important to understand that the FISH protocols disclosed herein can be performed entirely at about the physiological temperature of the body, e.g., of a mammal, preferably a human.

[0245] One advantage of the temperature range permitted by these methods is that microorganisms in clinical specimens can be studied under temperature conditions that approximate in vivo conditions, thus avoiding the effect of heat promoting the differential growth of one organism that would not otherwise be clinically significant while suppressing the appearance of another. For example, if a person suffers from a urinary tract infection in which the primary underlying irritant is Proteus mirabilis, and a clinical test is performed that involves heating the urine specimen, the clinical test will not prescribe the appropriate treatment to the clinician if heat promotes the growth of an otherwise minimal number of cells of Streptococcus agalactiae. The test will misidentify the microorganism that needs to be treated. To avoid such an outcome, the present disclosure provides compositions, devices and methods that allow FISH to be performed at constant physiological temperatures, which have particular utility in identifying microorganisms.

[0246] FIG. 7 shows how the steps of method 601 proceed. The specimen may include a mixture of cells 701 and one or more target cells 723 in the cells 701. The cells are incubated with probe 707. Preferably, a permeabilizing agent is used to permeabilize 625 the cells 701 including the target cells 723 to allow the label to diffuse therein. In a preferred embodiment, the label 707 specifically binds to the nucleic acid 715 target in the target cells 723. The cells are separated 629 from the label and the portion including the cells is examined to detect 635 the label. As discussed herein, the label is preferably a probe, for example, a fluorescently labeled oligonucleotide (e.g., about 10-18 bases long). The cells may be separated 629 from the unbound probe 707 by using magnetic particles that bind to the cells 701 and drawing the cells using a magnetic field B through a density medium leaving behind the unbound probe. Detection 625 may be performed by imaging (e.g., with a microscope) the separated cells. Cells may be imaged in or under a density medium, which may also be called a dye cushion, and may further contain a dye or pigment that prevents light from unbound probes from reaching the imaging device, such that any light spots in the image indicate the presence of a target cell 723 having a fluorescently labeled oligonucleotide probe hybridized to the target nucleic acid 715 therein.

[0247] FIG. 8 shows a permeabilizing agent 801 used to permeabilize 625 cells 701 according to the methods herein. In the embodiment shown, the agent 801 comprises a mixture of 3-([3-cholamidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate (sold under the name CHAPSO by Millipore Sigma, St. Louis, Mo.) and sulfobetaine 3-12 (available as SB3-12 from B-Biosciences, St. Louis, Mo.). These detergents permeabilize 625 the cells 701 to allow the probe 707 to enter and bind to the microbial target nucleic acid 715. Any suitable probe may be used in the methods herein. The incubating step may include exposing the cells to a reagent that permeabilizes the cells, thus allowing the label to enter the cells and bind to the target therein.

[0248] When a specimen containing cells 701 is delivered into the reagent well 109, the permeabilizing agent 801 facilitates the entry of the probe 707 into the microorganism (e.g., target cell 723) while the specimen is maintained at a temperature below about 40° C. The probe 707 may include a fluorescently labeled oligonucleotide 901 complementary to a segment of ribosomal RNA of a particular bacterial species. Preferably, the permeabilizing agent 801 includes one or more detergents (e.g., CHAPSO, SB3-12, TRITON® X100).

[0249] As shown, the probes 707 and permeabilizing agent 801 are provided in lyophilized beads 141 that are rehydrated and dissolved upon delivery of the analyte into the incubation well.

[0250] Thus, the method includes the steps of introducing a species-specific label (e.g., a fluorescently labeled oligonucleotide complementary to RNA in a target cell) into the specimen, optionally permeabilizing the cells with an agent (e.g., a detergent, such as 3-([3-cholamidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate (also known as CHAPSO); sulfobetaine 3-12 (also known as sb3-12); polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether (also known as TRITON® X100); nonylphenoxypolyethoxylethanol (also known as NP-40); others; or some combination thereof); separating unbound label from cells in the specimen; and imaging the cells to detect the label. The method is useful for testing specimens containing microorganisms, e.g., clinical specimens (e.g., to test for or detect causative agents of UTIs). When performed according to the present disclosure, the method provides FISH, which can be at any desired temperature, e.g., variable or constant physiological temperature.

[0251] Fluorescence in situ hybridization has been proposed for applications such as gene mapping and diagnosis of chromosomal abnormalities. See Nature Methods 2(3):237 (2005). These protocols involve hybridization of a biotin or digoxigenin labeled probe to denatured chromosomal DNA and detection of the probe using a fluorescent dye-conjugated reagent. Generally, these protocols require a denaturing step in which the probe itself and the target DNA are separately denatured at 70-80°C prior to probe hybridization, incubation and visualization. The disclosed method does not require a heating step and does not require any part of the specimen or reagents to be heated to about 70°C or even above 40°C. One important feature that provides a possible temperature range for the disclosed method involves the use of a permeabilizing agent (rather than heat) to deliver the probe 707 into the cell 701.

[0252] Suitable probes for use in the methods herein may include nucleic acid probes including DNA, RNA, peptide nucleic acid, modified bases, conformationally constrained nucleic acid, or combinations thereof. Suitable probes may include antibodies or antigens, binding molecules such as mannose-binding lectins or other collectins. Molecular or chemical structures and compositions such as polyethylene glycol, dyes, stains, intercalating dyes, crystal violet, safranine / carbol fuchsin, or any other composition or structure that can specifically bind to a target. In a preferred embodiment, the probe 707 comprises an oligonucleotide.

[0253] FIG. 9 shows a probe 707 according to a preferred embodiment. In a preferred embodiment, the probe 707 comprises an oligonucleotide 901 having a length between about 8 and 22 bases, preferably between about 10 and 22 bases. The probe preferably comprises DNA bases to avoid autocatalysis catalyzed by nucleophilic attack by the free electron of the 2' hydroxyl group (although RNA bases may be used or included as appropriate). The probe preferably has a melting temperature of about 45° C., e.g., between 40 and 50° C. Each oligonucleotide 901 is preferably labeled with at least one fluorophore 403. In a preferred embodiment, each oligonucleotide 901 also comprises one to several conformationally constrained nucleotides 905 (which may be variously referred to as locked or bridged nucleic acids). Thus, the probe 707 comprises a fluorescently labeled DNA oligo 901 having an optional conformationally constrained nucleic acid 905, and more preferably also at least one helper probe 907, optionally together with a second helper probe 913.

[0254] In certain embodiments, the label or probe 707 comprises a probe oligonucleotide 901 complementary to a microbial RNA. For the probe oligonucleotide 901 complementary to a microbial ribosomal RNA, the oligonucleotide 901 preferably has a length generally between 10 nt and 18 nt. The Tm is approximately 45° C. They are designed by looking at the structure of the rRNA. The helper probes disrupt the ribosomal structure. One reason for targeting rRNA is that the copy number is very high. There are several thousand copies per cell, so a de facto signal amplification is obtained. Thus, a preferred embodiment of the method 101 uses a reagent that includes one or more detergents (e.g., one or more of CHAPSO and SB3-12) and uses a probe oligonucleotide that targets a microbial ribosomal RNA. Specifically, the probe is a fluorescently labeled probe oligonucleotide complementary to a segment of the ribosomal RNA that is unique to the target species of interest. Preferably, the fluorescently labeled probe oligonucleotide is between 10 and 18 bases long and includes at least one conformationally constrained nucleic acid. Also preferably, the probe oligo 901 is provided with at least one helper probe 907 and optionally a second helper probe 913 .

[0255] Figure 10 shows the secondary structure of small subunit ribosomal ribonucleic acid (ssrRNA) 215, specifically, E. coli 16s rRNA. Although other bacteria do not have exactly the same 16S rRNA as E. coli, the secondary structure of the ribosome is highly conserved (see Woese & Fox, 1977), and therefore most of the helices shown have readily identifiable homologs in other bacteria. Preferred targets within the 16S rRNA include h44; h27; h16; h17; h18; h25; h27; h9; h10; h13; h23; h19; and h43. See Fuchs, 2000, Unlabeled helper oligonucleotides increase the in situ accessibility to 16S rRNA of fluorescently labeled oligonucleotide probes, Appl Environ Microbiol 66(8):3603-7, incorporated by reference. Once a tentative probe design has been determined, specificity and comprehensiveness can be tested using online tools, such as SILVA, "high quality ribosomal RNA databases," available as a website supported by the German network for bioinformatics infrastructure. See Pruesse, 2007, SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB, Nucl Acids Res 35:7188-7196 and Quast, 2013, The SILVA ribosomal RNA gene database project: improved data processing and web-based tools, Nucl Acids Res 41 (D1):D590-D596, both of which are incorporated by reference.The probe oligo 901 preferably contains at least one bridge or locked nucleic acid. It is acceptable for the helper probes 907, 913 to have lower specificity (see SILVA tool) than the oligo 901. The helper probes 907, 913 may preferably be about 20 nt in length.

[0256] The secondary structure of ribosomal RNA is instructive to illustrate the principles of probe design as well as the design and role of helper probes. Preferably, when oligo 901 hybridizes to a segment of microbial ribosomal RNA, helper probe 907 and optional second helper probe 913 are oligonucleotides that bind to the ribosomal RNA at a location within 1-30 bases of the segment to which the fluorescently labeled probe oligonucleotide binds. For example, the helper probe may hybridize to the microbial ribosomal RNA immediately upstream and downstream of the hybridized probe oligo 901. In the absence of helper probes 907, 913, inaccessibility of the target site may present a problem for hybridization of the 16S rRNA with the oligonucleotide probe. Herein, unlabeled oligonucleotides (helpers 907, 913) that bind adjacent to the probe target site are used to increase weak probe hybridization signals. Helper probes may be used to enhance the fluorescent signal. See Fuchs, 2000, Unlabeled helper oligonucleotides increase the in situ accessibility to 16S rRNA of fluorescently labeled oligonucleotide probes, Appl Environ Microbiol 66(8):3603-7, incorporated by reference.

[0257] Considerations when choosing probe target sequence include determining the theoretical specificity and comprehensiveness of FISH probe, optimizing the location of LNA bases, and designing helper probes for specific probes. Many pathogen targets already have FISH probes (as published or shortened to accommodate the temperature of this disclosure) that have been shown to be specific that can be used. Many of them can be found in probeBase, an online resource for rRNA-targeted oligonucleotide probes. See Loy, 2007, probeBase--an online resource for rRNA-targeted oligonucleotide probes: new features 2007, Nucleic Acids Res 35: D800-D804 and Loy, 2003, probeBase--an online resource for rRNA-targeted oligonucleotide probes, Nucleic Acids Res 31:514-516, both of which are incorporated by reference. Note that FISH is usually performed at much higher temperatures, therefore probes from these sources may need to be shortened or modified for method 101. One can also use the online tool "DECIPHER" to input the genus and let the DECIPHER tool suggest regions on 16S that are specific to that genus. See Wright, 2014, Automated Design of Probes for rRNA-Targeted Fluorescence In Situ Hybridization Reveals the Advantages of Using Dual Probes for Accurate Identification, Applied Env Microbiology, incorporated by reference.

[0258] Whether starting from an online tool or manually designing probes, it may be beneficial to test alignments (e.g., probes against 16S rRNA pairwise sequence alignments created by ClustalW) and select regions where the target sequence matches (preferably has multiple matches) but other pathogens do not. It may be beneficial to test for inclusion and specificity. The Tm should be above 40°C (as the disclosed method works at 35°C). A higher melting temperature may be preferred, but how high it can be depends on how many mismatches are present in the off-target sequences. Probe oligos according to the present disclosure have melting temperatures between 40°C and 60°C (e.g., for a 10-18 nt long DNA probe complementary to helix h17 in 16S rRNA with 2 or 3 LNA bases). Mismatches in the middle are more discriminatory than mismatches at the ends. Order of strength of mismatches: (best to worst): G / T, G / G, A / G, A / A, T / T, A / C, T / C, C / C. Preferably, choose a region of the rRNA that is more accessible.

[0259] The probes 707 are used to label specific target microorganisms. Another part of the method 601 includes a step of separating 629 the cells 701 from unbound probes. Any suitable method or technique may be used to separate 629 the cells 701 from unbound probes. Suitable techniques for separating the cells 701 from unbound probes include centrifugation, flow cytometry, fluorescence activated cell sorting, column separation, digestion of unbound probes via one or more nucleases, etc., or combinations thereof. In a preferred embodiment, the cells 701 are separated 629 from unbound probes 707 by the use of magnetic particles. For example, the incubation 613 step may include exposing the cells to magnetic particles that bind to the surface of the cells.

[0260] Any suitable magnetic particle that binds to the surface of a cell may be used, including, for example, magnetic particles bound to antibodies, collagen-containing C-type lectins (also known as collectins, e.g., mannose-binding lectins), or chemical groups that bind to bacterial cells. In certain preferred embodiments, the magnetic particles preferably comprise PAA.

[0261] FIG. 11 shows a magnetic particle 1105 that includes a chemical group 1111 that binds to a bacterial cell surface. The chemical group 1111 may include, for example, diethylamine ethyl starch; dextran sulfate; polyaspartic acid; polyacrylic acid; polystyrene sulfonate; polydiallyldimethylamine; or combinations thereof. As shown, the magnetic particle 1105 includes a polyaspartic acid chemical group 1111. This particle is sold as fluidMAG-PAA by chemicell GmbH (Berlin, Germany). The fluidMAG-PAA particle is a polyaspartic acid that binds to the bacterial surface. The cells may be exposed to the magnetic particle 1105 in the presence of a compound 1121 that promotes the binding of the chemical group 1111 to the bacterial cell surface.

[0262] To effectively bind the particles 1105 to the cells 701, it may be beneficial to include an agent 1121 that promotes binding of the PAA to the cell surface. Any suitable agent 1121 may be included to promote binding. For example, in some embodiments, the agent includes a mixture of different quaternary ammonium salts including cetrimonium bromide (CTAB), also known as cetrimide. Cetrimide promotes binding of the PAA to the cell surface for magnetic capture, solving particular troubles with gram-positive organisms. Gram-negative organisms may be found to bind to the fluidMAG-PAA without trouble. If the target microorganism of interest is gram-positive, it may be preferable to include an agent 1121 (e.g., cetrimide). Thus, in a preferred embodiment of method 601, the label comprises a fluorescently labeled probe oligonucleotide 901 complementary to a ribosomal RNA 215 unique to that species; the incubating 613 step also comprises exposing the cells to magnetic particles 1105 that bind to the surface of the bacterial cells 701; and the separating 629 step comprises applying a magnetic field B to the cells 701.

[0263] FIG. 12 shows magnetic particle 1105-bound cells 701 being separated 629 from unbound probes 700 by drawing the cells 701 through a density gradient medium 1201 using an applied magnetic field B. The density medium 1201 may be provided in the depicted tube or imaging well 111 (which may contain dye to provide the "dye cushion 115") such that the separating 629 step may include distributing the magnetic particle 1105-bound cells 701 across the dye cushion 115 and drawing the bound cells 701 through the dye cushion 115 onto the imaging surface 1205 using a magnetic field B, leaving the unbound probes 700 on the surface of the dye cushion 115. The detecting 635 step may then include imaging the imaging surface 1205 with a fluorescent microscope, all steps may be performed at a temperature below 40° C. Preferably, these steps are performed at a temperature between about 36° C. and 39° C. Thus, as shown, incubating 613 includes exposing the cells to magnetic particles 1105 that bind to the surface of the bacterial cells, and separating 629 includes attracting the bound cells away from the unbound label using a magnetic field B. Preferably, separating 629 includes distributing the magnetic particle-bound cells across the surface of the dye cushion 115, and using a magnetic field to attract the bound cells through the dye cushion 115 and onto the imaging surface 1205, leaving the unbound label on the surface of the dye cushion.

[0264] As discussed, an embodiment of separation 629 uses a density gradient medium 1201 that may include a dye to provide a dye cushion. Thus, the dye cushion 115 is a material that includes a density gradient medium that further includes a dye.

[0265] The dye cushion 115 may be, for example, a density gradient medium (e.g., a solution of iodixanol or polyvinylpyrrolidone coated colloidal silica particles, optionally dried or lyophilized prior to exposure to the analyte) that further contains a dye that absorbs light from unbound probes 700. The cushion may include a high density material to exclude unselected components of the reaction from the detection zone. The cushion is generally a denser layer (liquid, or dried or lyophilized) than the reaction components. The cushion may include various density agents, alone or in combination (and at various concentrations), including, for example, sucrose, diatrizoate, iodixanol (also known as OptiPrep), NaCl, CsCl, Percoll, or albumin. Embodiments may also incorporate other density agents, such as other commonly used density agents such as iodixanol, sodium diatrizoate, sodium, sucrose, and other sugars, oligosaccharides, synthetic polymers (e.g., Ficoll), and salts such as cesium chloride, potassium bromide, and the like. Embodiments may use dyes to match different signaling features and moieties in use. For example, the dye Toluidine Blue O may be used with the fluorescent label Texas Red (sulforhodamine). One embodiment uses a 65 μL aliquot of dye cushion reagent that is 2 mg / mL Chromotrope R2 and 10% v / v OptiPrep (a 60% w / v solution of iodixanol) plus 5% w / v trehalose pipetted into the assay well. The dye cushion may be 15% OptiPrep and 5 mg / mL Chromotrope R2 pre-aliquoted into the imaging well 111 of the cartridge 101.

[0266] With respect to the imaging well 111, the dye cushion 115 may be formed by preparing a solution of iodixanol or polyvinylpyrrolidone containing any optional dyes and drying or lyophilizing the solution in the imaging well 111 to form the dye cushion 915. The dye cushion 915 then becomes essentially solid (e.g., dried, the imaging well 111 may be stored in any orientation, including upside down, until use). When a liquid specimen is delivered into the imaging well 111, the liquid rehydrates the dye cushion 115. Indeed, the reagents disclosed and discussed throughout this specification for use in the method 601 may be provided in a dried or lyophilized form, for example, for later use in a protocol for FISH at a constant physiological T. This allows the reagents to be prepared and loaded in a dry state onto a cartridge, which may then be shipped or stored and later used in the disclosed methods.

[0267] In a preferred embodiment, the cartridge 101 also includes a dye cushion 115 adjacent to the magnetic particles 1105 that bind to the bacterial cell surface; and a transparent wall that provides an imaging surface 1205. When a magnetic field is applied across the dye cushion 115, the magnetic field attracts the magnetic particles 1105 through the dye cushion and into the transparent wall. Preferably, the magnetic particles 1105 (and any compounds 621 that facilitate binding) are also included in the lyophilized beads 141. The dye cushion 115 includes a solution of density gradient medium 1201 that further includes a dye that absorbs light from unbound probes 700. In the illustrated embodiment, the dye cushion 115 and the transparent wall 1205 are provided in an imaging well 111 in fluid communication with the reagent well 109. The dye cushion 115 may be provided as a gel or in a dried or lyophilized state in the imaging well within the cartridge until it is wetted by the specimen.

[0268] Preferably, the magnetic particles 1105 in the lyophilized beads 141 include a chemical group that binds to the bacterial cell surface, and the cartridge further includes a compound that facilitates the binding of the chemical group to the bacterial cell surface. The compound that facilitates the binding of the chemical group to the cell surface can be cetrimide, and the chemical group can be, for example, diethylamine ethyl starch; dextran sulfate; polyaspartic acid; polyacrylic acid; polyglutamic acid; polystyrene sulfonate; or polydiallyldimethylamine.

[0269] The probe 707 may be provided as a fluorescently labeled oligonucleotide 901 complementary to a segment of ribosomal RNA 215 of a particular bacterial species in lyophilized beads 141, which also preferably contain at least one helper probe oligonucleotide that binds to the ribosomal RNA at a location within 1-30 bases of the segment. The fluorescently labeled oligonucleotide 901 may be between 10 and 18 bases in length, and includes at least one conformationally constrained nucleic acid for use in, for example, FISH, which in some embodiments is at a constant physiological temperature.

[0270] The reagent composition, probes, helper probes and compounds are provided as lyophilized beads 141 that are rehydrated and dissolved by delivery of the specimen into the cartridge 101. The dye cushion 115 includes a density gradient medium 1201 that further includes a dye that absorbs light from unbound probes. The dye cushion 115 may be provided in a dried or lyophilized state in an imaging well within the cartridge until wetted by the specimen. The method 601 and cartridge 101 may be used to perform antibiotic susceptibility testing.

[0271] FIG. 13 shows a workflow in which FISH at physiological temperature is used to perform antibiotic susceptibility testing (AST). A specimen is loaded into the specimen well 103 of the cartridge 101. The divided wells 105 contain antibiotics, either different antibiotics or different concentrations of antibiotics. One "channel" may contain no antibiotics as a control or to establish a baseline of growth. The cartridge is connected to a source of air pressure, a gate switch, and a fluorescent microscope or similar imaging instrument. The gate switch slides a sliding valve to a first position, and the specimen well 103 is in fluid communication with the divided wells 105. Air pressure is applied through a fitting and the specimen is divided between the divided wells. Herein, the specimen may be incubated with multiple antibiotics distributed across the divided wells 105.

[0272] The sliding valve 107 is slid to a third position, where the divided wells 105 are in fluid communication with the reagent wells 109. Air pressure is applied and an aliquot of specimen is delivered from the divided wells 105 to the respective reagent wells 109. In each reagent well 109, the specimen aliquot is incubated with a probe specific to the target nucleic acid of the microorganism species as well as magnetic particles at no more than 45° C. The sliding valve 107 is slid to a second position, where the reagent wells are sealed. The specimen is delivered to the imaging wells 111 and a magnetic field is applied to separate intact cells in the specimen from unbound probe. This may be performed by sliding the cartridge onto a magnet. Field B attracts the cells onto the imaging surface. The bound probes within the intact cells are detected to quantify the growth of the species in each aliquot specimen. Growth or lack thereof may be inversely correlated with which antibiotic was present in which divided well 105. For those imaging wells 111 in which no pathogen growth is detected (eg, fluorescence microscopy shows no fluorescence), the specimen is indicated to be susceptible to the antibiotic present in the corresponding divided well 105 .

[0273] An advantage of the cartridges 101 is that they are suitable for use with instruments that interact with the cartridges 101 to automate steps.

[0274] Detectable labels may include fluorescent molecules, radioisotopes, mass tags for mass spectrometry, or chemiluminescent molecules. Detectable labels may include target-specific moieties to preferentially bind to target cells or microorganisms. Target-specific binding molecules may include, for example, antibodies to target-specific molecules or antigens, or oligonucleotide or polynucleotide probes complementary to target-specific nucleic acid sequences. In a preferred embodiment, the detectable labeling of microorganisms constitutes fluorescent in situ hybridization (FISH). FISH analysis uses fluorescent probes that include a nucleic acid (or nucleic acid analog) probe portion and a fluorophore probe portion that binds to a target-specific nucleic acid sequence via reassociation, so that the target can then be optically detected. For example, a probe targeting a target-specific 16S rRNA can be used to selectively tag and detect microorganisms. 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 assays, several distinct target-specific fluorescent probes can be used to independently identify multiple distinct categories of targets in a single specimen. The distinct fluorescent probes can include distinct nucleic acid probe portions designed such that under assay reassociation conditions, the nucleic acid probe portions of the fluorescent probes preferentially reassociate with target-specific cellular nucleic acid sequences for distinct categories of target cells or microorganisms. A detailed discussion of probe reassociation can be found in U.S. Patent Application Publication No. 20030228599, which is incorporated herein by reference.

[0275] The fluorophores on the separate fluorescent probes may have separate photonic signals, so that the fluorescent signals for different categories of target cells or microorganisms in the assay can be distinguished by their separate photonic signals. Imaging methods for distinguishing multiple separate photonic 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, a single specimen portion can be tested for the presence of multiple specific target cells or microorganisms in a single processing and imaging well.

[0276] In certain embodiments, FISH analysis for identification and / or quantification of target cells or microorganisms in a specimen can be performed isothermally, without reagent exchange, without cell fixation, allowing for automated on-device processing with a time between initiating the reaction and obtaining imaging results of about 30 minutes or less.

[0277] The FISH analysis described herein can be performed at a constant physiological temperature so that several cartridges can be maintained at the same temperature for incubation and FISH analysis, thereby requiring only a single temperature for the interior of the instrument and avoiding the need for separate incubation chambers maintained at different temperatures.

[0278] Generally, physiological temperature refers to the body temperature of an organism, such as an animal. The process steps, molecular species, and chemical reagents disclosed herein are useful for hybridizing fluorescent probes to nucleic acids in cells and imaging those probes at physiological temperatures without lysing the cells. There is flexibility regarding the temperature to which the specimen is exposed and the steps are performed. The disclosed methods may be usefully performed at temperatures that fluctuate but do not exceed 45°C, and may even operate at temperatures that do not exceed 40°C. The disclosed methods and compositions are useful and functional when used at temperatures within the range of, for example, 36-39°C. In fact, the disclosed methods may be performed on equipment that maintains temperatures at or about human body temperature, i.e., about 37°C for healthy humans, 38°C for humans with fever, or 36°C for some nocturnal human temperature fluctuation patterns. By "about" we mean within a decimal point, etc., i.e., 36.3 is considered to be about 36.5, and 37.7 is considered to be about 37.5. It is important to understand that the FISH protocols disclosed herein can be performed entirely at about the physiological temperature of the body, e.g., of a mammal, preferably a human.

[0279] One advantage of the temperature ranges permitted by these methods is that microorganisms in clinical specimens can be studied under temperature conditions that approximate in vivo conditions, thus avoiding the effect of heat promoting the differential growth of one organism that would not otherwise be clinically significant while suppressing the appearance of another. For example, if a person suffers from a urinary tract infection in which the primary underlying irritant is Proteus mirabilis, and a clinical test is performed that involves heating the urine specimen, the clinical test will not prescribe the appropriate treatment to the clinician if heat promotes the growth of an otherwise minimal number of cells of Streptococcus agalactiae. The test will misidentify the microorganism that needs to be treated. To avoid such an outcome, the present disclosure provides compositions, devices and methods for performing FISH that can be performed at constant physiological temperatures, which have particular utility in identifying microorganisms.

[0280] A species-specific label (e.g., a fluorescently labeled oligonucleotide complementary to RNA in a target cell) can be introduced into the specimen, and the cells can be permeabilized as necessary using an agent (e.g., a detergent, such as 3-([3-cholamidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate (also known as CHAPSO); sulfobetaine 3-12 (also known as sb3-12); polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether (also known as TRITON® X100); nonylphenoxypolyethoxylethanol (also known as NP-40); others; or some combination thereof). The labeled species can be separated from unbound label in the specimen; and the cells can be imaged to detect the label. Such methods are useful for testing specimens containing microorganisms, e.g., clinical specimens (e.g., to test for or detect causative agents of UTIs). When practiced in accordance with the present disclosure, the methods allow for the performance of FISH, which can be performed at any variable temperature, for example, constant physiological temperature.

[0281] Fluorescence in situ hybridization has been proposed for applications such as gene mapping and diagnosis of chromosomal abnormalities. See Nature Methods 2(3):237 (2005). These protocols involve hybridization of biotin or digoxigenin labeled probes to denatured chromosomal DNA and detection of the probe using fluorescent dye-conjugated reagents. Generally, these protocols require a denaturing step in which the probe itself and the target DNA are separately denatured at 70-80°C prior to probe hybridization, incubation and visualization. The disclosed method does not require a heating step and does not require any part of the specimen or reagents to be heated to about 70°C or even above 40°C. One important feature that provides a possible temperature range for the disclosed method involves the use of a permeabilizing agent (rather than heat) to deliver the probe into the cell.

[0282] The permeabilizing agent may include a mixture of 3-([3-cholamidopropyl]dimethyl-ammonio)-2-hydroxy-1-propanesulfonate (sold under the name CHAPSO by Millipore Sigma, St. Louis, MO) and sulfobetaine 3-12 (available as SB3-12 from B-Biosciences, St. Louis, MO). These detergents permeabilize the cells to allow the probe to enter so as to bind to the microbial target nucleic acid. Any suitable probe may be used in the methods herein.

[0283] Suitable probes for use in the methods herein may include nucleic acid probes including DNA, RNA, peptide nucleic acid, modified bases, conformationally constrained nucleic acid, or combinations thereof. Suitable probes may include antibodies or antigens, binding molecules such as mannose-binding lectins or other collectins. Molecular or chemical structures and compositions such as polyethylene glycol, dyes, stains, intercalating dyes, crystal violet, safranine / carbol fuchsin, or any other composition or structure that can specifically bind to a target. In a preferred embodiment, the probe 707 comprises an oligonucleotide.

[0284] The probes may comprise oligonucleotides having a length between about 8 and 22 bases, preferably between about 10 and 22 bases. The probes preferably comprise DNA bases to avoid autocatalysis catalyzed by nucleophilic attack by the free electrons of the 2' hydroxyl group (although RNA bases may be used or included as appropriate). The probes preferably have a melting temperature of about 45°C, e.g., between 40 and 50°C. Each oligonucleotide is preferably labeled with at least one fluorophore. In a preferred embodiment, each oligonucleotide also comprises one to several conformationally constrained nucleotides (variously referred to as locked or bridged nucleic acids). Thus, the probes comprise fluorescently labeled DNA oligos with optional conformationally constrained nucleic acids, and more preferably at least one helper probe as well, optionally with a second helper probe.

[0285] In certain embodiments, the label or probe comprises a probe oligonucleotide complementary to microbial RNA. For the probe oligos complementary to microbial ribosomal RNA, the oligos generally have a length between 10nt and 18nt. The Tm is approximately 45°C. Helper probes can be used to disrupt ribosomal structure. One reason for targeting rRNA is that the copy number is very high compared to messenger and transfer RNA in bacterial cells. Since there are thousands of copies per cell, linking a signaling molecule such as a fluorophore to the rRNA targeting probe results in a virtual signal amplification with thousands of targets in a single cell. Thus, picking out a single cell in subsequent fluorescence imaging is easier considering the signal-to-noise ratio obtained by such a high concentration of labels in the target bacterial cell. Thus, a preferred embodiment of the method uses a reagent that includes one or more detergents (e.g., one or more of CHAPSO and SB3-12) and uses a probe oligonucleotide that targets microbial ribosomal RNA. Specifically, the probe can be a fluorescently labeled probe oligonucleotide that is complementary to a segment of ribosomal RNA that is unique to the target species of interest.Preferably, the fluorescently labeled probe oligonucleotide is between 10 and 18 bases long and contains at least one conformationally constrained nucleic acid.Also preferably, the probe oligo is provided with at least one helper probe and optionally a second helper probe.

[0286] Preferably, when the oligo hybridizes to a segment of microbial ribosomal RNA, the helper probe and any optional second helper probe are oligonucleotides that bind to the ribosomal RNA at a location within 1-30 bases of the segment to which the fluorescently labeled probe oligonucleotide binds. For example, the helper probe may hybridize to the microbial ribosomal RNA immediately upstream and downstream of the hybridized probe oligo. In the absence of a helper probe, inaccessibility of the target site may present a problem for hybridization of the 16S rRNA with the oligonucleotide probe. In certain embodiments, unlabeled oligonucleotides that bind adjacent to the probe target site are used to increase weak probe hybridization signals. Helper probes may be used to enhance fluorescent signals. See Fuchs, 2000, Unlabeled helper oligonucleotides increase the in situ accessibility to 16S rRNA of fluorescently labeled oligonucleotide probes, Appl Environ Microbiol 66(8):3603-7, incorporated by reference. The considerations when choosing probe target sequence include determining the theoretical specificity and comprehensiveness of FISH probe, optimizing the location of LNA base, and designing helper probe for specific probe.Many pathogen targets already have FISH probes (as published or shortened to adapt to the temperature of this disclosure) that have been shown to be specific and can be used.Many of them can be found in probeBase, an online resource for rRNA-targeting oligonucleotide probes.See Loy, 2007, probeBase--an online resource for rRNA-targeted oligonucleotide probes: new features 2007, Nucleic Acids Res 35: D800-D804 and Loy, 2003, probeBase--an online resource for rRNA-targeted oligonucleotide probes, Nucleic Acids Res 31:514-516, both of which are incorporated by reference. Note that conventional FISH reactions are usually performed at temperatures much higher than those used in the preferred constant physiological temperature method. Thus, probes from conventional sources may need to be shortened or modified for use in the methods described herein. The online tool "DECIPHER" can also be used to input a genus and the DECIPHER tool can suggest regions on 16S that are specific to that genus. See Wright, 2014, Automated Design of Probes for rRNA-Targeted Fluorescence In Situ Hybridization Reveals the Advantages of Using Dual Probes for Accurate Identification, Applied Env Microbiology, incorporated by reference. Whether starting with an online tool or manually designing probes, it may be beneficial to test alignments (e.g., probes against 16S rRNA pairwise sequence alignments created by ClustalW) and select regions where the target sequence matches (preferably has multiple matches) but other pathogens do not. It may be beneficial to test inclusion and specificity. The Tm should be above 40°C (as the disclosed method works at 35°C). A higher melting temperature may be preferred, but how high it can be depends on how many mismatches are present in the off-target sequence.Probe oligos according to the present disclosure have melting temperatures between 40°C and 60°C (e.g., for a 10-18 nt long DNA probe complementary to helix h17 in 16S rRNA with 2 or 3 LNA bases). Central mismatches are more discriminatory than terminal mismatches. Order of mismatch strength: (best to worst): G / T, G / G, A / G, A / A, T / T, A / C, T / C, C / C. Preferably, choose a region of the rRNA that is more accessible.

[0287] Once a tentative probe design is decided, specificity and comprehensiveness can be tested using online tools, such as SILVA, "high quality ribosomal RNA databases," available as a website supported by the German network for bioinformatics infrastructure. See Pruesse, 2007, SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB, Nucl Acids Res 35:7188-7196 and Quast, 2013, The SILVA ribosomal RNA gene database project: improved data processing and web-based tools, Nucl Acids Res 41 (D1):D590-D596, both of which are incorporated by reference. The probe oligo preferably contains at least one bridge or locked nucleic acid. It is acceptable for the helper probe to have a lower specificity than the oligo (see SILVA tool). The helper probe may preferably be about 20 nt long.

[0288] After the target cells are labeled, the cartridges and devices described herein are operable to separate the labeled cells from unbound labels for imaging to reduce background signals. Any suitable method or technique can be used to separate the cells from unbound probes. Suitable techniques for separating the cells from unbound probes include centrifugation, flow cytometry, fluorescence activated cell sorting, column separation, digestion of unbound probes via one or more nucleases, etc., or combinations thereof. Because these methods are performed in a closed cartridge, traditional washing steps to remove unbound labels are difficult to achieve. In a preferred embodiment, the cells are separated from unbound probes by the use of magnetic particles. For example, the incubation step can include exposing the cells to magnetic particles that bind to the surface of the cells.

[0289] The magnetic particles may contain chemical groups that bind to bacterial cell surfaces. The chemical groups may include, for example, diethylamine ethyl starch; dextran sulfate; polyaspartic acid; polyacrylic acid; polystyrene sulfonate; polydiallyl dimethylamine; or combinations thereof. Such particles are, for example, sold as fluidMAG-PAA by chemicell GmbH (Berlin, Germany). The fluidMAG-PAA particles are polyaspartic acid that bind to bacterial surfaces. The cells may be exposed to the magnetic particles in the presence of a compound that promotes the binding of the chemical groups to the bacterial cell surface.

[0290] To effectively bind the particles to the cells, it may be beneficial to include an agent that promotes the binding of the PAA to the cell surface. Any suitable agent may be included to promote the binding. For example, in some embodiments, the agent includes a mixture of different quaternary ammonium salts, including cetrimonium bromide (CTAB), also known as cetrimide. Cetrimide promotes the binding of the PAA to the cell surface for magnetic capture, solving a particular problem with gram-positive organisms. Gram-negative organisms may be found to bind to fluidMAG-PAA without any trouble. If the target microorganism of interest is gram-positive, it may be preferable to include an agent (e.g., cetrimide). Thus, in a preferred embodiment of the method, the label includes a fluorescently labeled probe oligonucleotide that is complementary to ribosomal RNA that is unique to that species; the incubating step also includes exposing the cells to magnetic particles that bind to the surface of the bacterial cells; and the separating step includes applying a magnetic field B to the cells.

[0291] The magnetic particle-bound cells can be separated from the unbound probes by drawing the cells through a density gradient medium using an applied magnetic field, such as provided by a magnetic subsystem or station in the instrument. The density medium can be provided in a tube or well (which can contain a dye to provide a "dye cushion") so that the separating step can include distributing the magnetic particle-bound cells across a dye cushion, and using a magnetic field to draw the bound cells through the dye cushion onto an imaging or detection surface, leaving the unbound probes on the surface of the dye cushion. The detecting step can then include imaging the imaging surface with a fluorescent microscope, and all steps can be performed at a temperature below 40°C. Preferably, these steps are performed at a temperature between about 36°C and 39°C.

[0292] As discussed, embodiments of the separations use a density gradient medium that may include a dye to provide a dye cushion. The dye cushion is thus a material that includes a density gradient medium that further includes a dye.

[0293] The dye cushion can be, for example, a density gradient medium (e.g., a solution of iodixanol or polyvinylpyrrolidone coated colloidal silica particles, optionally dried or lyophilized prior to exposure to the analyte) that further contains a dye that absorbs light from unbound probe. The cushion can include a high density material to exclude unselected components of the reaction from the detection zone. The cushion is a layer (liquid, or dried or lyophilized) that is generally denser than the reaction components. The cushion can include various density agents, alone or in combination (and at various concentrations), including, for example, sucrose, diatrizoate, iodixanol (also known as OptiPrep), NaCl, CsCl, Percoll, or albumin. Embodiments can also incorporate other density agents, including other commonly used density agents, such as iodixanol, sodium diatrizoate, sodium, sucrose, and other sugars, oligosaccharides, synthetic polymers (e.g., Ficoll), as well as various salts, such as cesium chloride, potassium bromide, and the like. Embodiments may use dyes to match different signaling features and moieties in use. For example, the dye Toluidine Blue O may be used in conjunction with the fluorescent label Texas Red (sulforhodamine).

[0294] The dye cushion can be formed by preparing a solution of iodixanol or polyvinylpyrrolidone containing any optional dyes and drying or lyophilizing the solution in the imaging well of the cartridge. The dye cushion then becomes essentially solid (e.g., dried, the well can be stored in any orientation, including upside down, until use). When a liquid specimen is delivered into the well, the liquid rehydrates the dye cushion. Indeed, the reagents disclosed and discussed throughout this specification for use in the method can be provided in a dried or lyophilized form for later use in a protocol for FISH at a constant physiological temperature or a cyclical temperature that may be below or above physiological temperature. This allows the reagents to be prepared and loaded in a dry state onto the cartridge, which can then be shipped or stored and later used in the disclosed method.

[0295] The magnetic particles, detectable labels and target-specific binding molecules preferably form complexes with any targets present in the specimen. The magnetic particles and applied magnetic field can be used to physically separate the bound detectable labels from the unbound detectable labels in the solution without a washing step. The dye cushion layer described in US Pat. No. 9,643,180 can be used in conjunction with the magnetic particles and magnetic field to attract the labeled target cells or microorganisms through the high-density dye layer and deposit them on the detection surface in the well of the test device for imaging analysis. The dyes in the dye cushion layer are preferably selected to absorb the excitation and emission light used by the instrument for imaging. Thus, the signal from the unbound labeling moieties in the assay layer does not significantly interfere with the detection of the signal from the magnetically deposited labeled target cell or microorganism complex on the detection surface. Similarly, the use of the dye cushion prevents any autofluorescence from the specimen matrix, also included in the assay layer, from significantly interfering with the detection of the signal from the deposited labeled target cell complex. These properties of the dye cushion may allow for detection of target cells or microorganisms without sample preparation by the user and without a washing step to remove unbound label from the test device.

[0296] Digital imaging of labeled target cells or microorganisms can be achieved using a digital imager. In the preferred case of fluorescent labeling, various lenses, illumination sources, excitation light sources and filters can be used. The imaging module can include any device capable of generating digital images of detectably labeled target cells 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.

[0297] Imaging may be performed under a single set of conditions, or the light source, filters and / or lenses may 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 may allow for the observation and enumeration of individual bacteria or other target cells.

[0298] 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.

[0299] 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).

[0300] 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.

[0301] 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.

[0302] The analysis cartridge 101 has an identifier, such as a barcode sticker, which when analyzed or read by the instrument or a reader in the instrument, associates the cartridge with a set of instructions for processing within the instrument and may contain information regarding the source of the specimen for an associated assay result by a particular patient. EXAMPLES

[0303] Example 1 Limits of detection (LoD) for Gram-negative bacteria using a novel rapid fluorescent in situ hybridization assay

[0304] 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:

[0305] 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.

[0306] 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 ...

[0307] Preparation of FISH probes: Two species-specific DNA oligomer sets for E. coli and K. pneumoniae and one species-specific DNA oligomer set for P. aeruginosa were heated in a water bath between 80-85°C for 10 min and then placed on ice to reduce aggregation. The DNA oligomer sets included species-specific DNA oligonucleotides labeled with a fluorescent dye (Alexa647N, Thermo Fischer) either at the 5' end or at both the 5' and 3' ends of the oligonucleotide, as well as two to six helper oligonucleotides that bind adjacent to or near the specific probes and are designed to disrupt the local secondary structure of the ribosomal subunits and allow for higher hybridization efficiency of the labeled specific probes to the target rRNA. The probe sequences used in this example are shown in Table A in Figure 17.

[0308] 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).

[0309] 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".

[0310] 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.

[0311] Data analysis: The number of fluorescent objects detected at each cell concentration was determined. Data from all eight cell concentrations were fitted to a linear regression line and the slope, intercept and standard deviation of the lowest three cell inputs were used to determine the upper limit of blank (LoB) and limit of detection (LoD) for each bacterium tested. result:

[0312] All three bacteria tested showed low limits of detection. Figures 14, 15 and 16 show data generated for E. coli, K. pneumoniae and P. aeruginosa using the linear fits used to calculate the LoB and LoD. The LoB and LoD are shown in terms of detectable CFU in a single reaction well.

[0313] Conclusion: The novel and rapid FISH method described in this example is shown to be a highly sensitive method, with a limit of detection of approximately 500 CFU or less per reaction, using minimally processed urine matrix.

[0314] Variations. This example is illustrative of the performance of this novel FISH method and is not limited to the specific details included in the description. Thus, those skilled in the art will easily understand that many variations are possible, including using different probe sequences and nucleic acid structures (PNA, LNA, etc.), alternative assay chemistries (different detergents, chaotropes, fluorophores, buffers, pH, temperature, reaction time, component concentrations), urine concentrations and urine processing procedures. This methodology can obviously be extended to other biological specimens and other bacterial and non-bacterial pathogens.

[0315] Figure 14 shows the limit of detection (LoD) for E. coli ATCC 19138. 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.

[0316] Figure 15 shows the limit of detection (LoD) for P. aeruginosa ATCC 9721. 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.

[0317] Figure 16 shows the limit of detection (LoD) for K. pneumoniae ATCC 700603. 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.

[0318] FIG. 17 is a table of the probe sequences used in this example.

[0319] Example 2 Comprehensiveness: Detecting and identifying different strains of bacterial species using the rapid FISH method of the present invention

[0320] Overview. This example demonstrates the use of the present invention to detect different strains of targeted bacterial species. Raw data for 11 different E. coli strains is shown, and data for K. pneumoniae, P. aeruginosa, P. mirabilis and Enterococcus spp. are summarized. Bacterial cell targets were labeled in 30 minutes using isothermal fluorescence in situ hybridization (FISH) and detected with a MultiPath™ CCD camera-based detection system. Experimental method.

[0321] Bacterial cell preparation: Bacterial cultures for the different strains were obtained by inoculating Trypticase Soy Broth (TSB, Hardy Diagnostics catalog U65) with 3-5 colonies from a fresh Tryptic Soy Agar plate (TSA, BD catalog 221185) and growing at 35°C for 1.5-3 hours to achieve log phase growth. Using optical density at 600 nm to estimate cell concentration, cells were diluted in 1x cation adjusted Mueller-Hinton Broth (MHBII, Teknova catalog M5860) to approximately 600 and 3000 CFU per reaction. For more accurate percent cell detection calculations, these estimated bacterial inputs were adjusted using colony counts. Plate counts were determined by diluting log phase cultures in MHBII to approximately 500 CFU / mL, plating 100 μL onto TSA plates, and counting colonies after 16-24 hours of growth at 35°C.

[0322] Preparation of magnetic particles: Polyaspartic acid conjugated magnetic particles (Fluidmag-PAA, Chemicell, catalog 4108), used for non-specific capture of bacterial cells, were diluted to 2.75 × 10 in 50 mM EPPS buffer, pH 8.2. 12Fluorescent magnetic microspheres containing green dye (Dragon Green Fluorescent Microspheres, BANGS Laboratories, catalog MEDG001) were diluted 1:20 to 3 × 10 6 The magnetic particles were added to the suspension at a final concentration of 10 ...

[0323] Labeling of bacterial cells: A 100 μL labeling reaction was prepared by mixing diluted cells, isothermal hybridization buffer (0.9× MHBII, 3× SSC (1.5 M NaCl, 0.15 M sodium citrate, Sigma, catalog S6639), 0.77% w / v CHAPSO (Sigma catalog C3649), 0.72% w / v SB3-12 (Sigma catalog D0431), 0.13 M guanidine thiocyanate (Sigma catalog G9277), 0.18% w / v cetrimide (Sigma catalog M7365)), species-specific Alexa647N-labeled DNA or LNA-containing DNA probes targeted to 16S or 23S bacterial rRNA (Integrated DNA Technologies, IDT), helper probes to promote efficient hybridization (IDT) and 30 μL of pooled human urine (Innovative The probe sequences are shown in the table in FIG.

[0324] Urine was first processed through a Zeba 7K MWCO spin column (Thermo Fisher, catalog 89893 or 89892 depending on urine volume) according to the manufacturer's instructions. 10 μL of magnetic particle preparation was then added to this mixture. The final reaction mixture was transferred to a microtiter plate containing 50 μL (pre-dried) "dye cushion" (50 mM TRIS pH 7.5 (Teknova catalog T1075), 7.5% v / v Optiprep (Sigma catalog D1556), 50 mg / mL Direct Black 19 (Orient catalog 191L)) and incubated at 35° C. for 30 minutes to allow simultaneous rehydration of the "dye cushion", labeling of bacterial cells, and binding of magnetic particles to the bacterial cell surface. After incubation, the microtiter plate was placed on a magnetic field (Dexter magnetic technologies, catalog 54170260) for 4 minutes to bring the magnetic particles, the fraction containing the labeled cells, into close proximity with the imaging surface at the bottom of the wells via a "dye cushion."

[0325] Imaging of Labeled Cells: Labeled bacterial cells on the MultiPath Laboratory Imaging System is a custom-built instrument and software capable of automatically capturing image data from selected wells of a microtiter plate. It uses a high-precision linear stage from Prior Scientific (Rockland, MA) to position each well on a fluorescence-based image acquisition subsystem. The instrument is capable of imaging in four separate color channels and uses an objective lens, illumination LEDs, a fluorescence filter set, and a camera. The objective lens has a field of view designed to capture an image of the entire microtiter plate well. The illumination module light source consists of two high-power LEDs per color channel. A series of fluorescent image frames are captured by the camera using a 3.1MP Sony IMX265 monochrome sensor with 12 bits per pixel quantization. A final image for each well is then formed by summing multiple frames. 16 frames were captured with a 100 ms exposure using a 635 / 25 nm excitation and 680 / 40 nm emission filter. The focal particle is imaged with 470 / 40 nm excitation and 520 / 40 nm excitation filters, capturing 2 frames with a 20 ms exposure.

[0326] Data analysis: For each bacterium, the number of fluorescent objects was determined (assay signal). A bacterial strain was considered detected if a signal was detected that was greater than 3 standard deviations above the signal in the cell-free condition.

[0327] Results. Figure 105 shows the assay signal for eleven E. coli strains. All eleven strains were detected above background "no cells" conditions for a cell input of approximately 600 CFU per assay. Figure 18 shows the percentage of cells detected (total assay signal in cell input wells - background assay signal / total cell input). * Data presented are presented as percentile (%) = 100. Although detection efficiency was somewhat variable from strain to strain, this did not inhibit the ability of the assay to detect each of the 11 different E. coli strains.

[0328] The table in Figure 19 summarizes the comprehensiveness results for E. coli, K. pneumoniae, P. aeruginosa, P. mirabilis and Enterococcus spp., which were analyzed in the same manner as E. coli. The strains tested for K. pneumoniae were ATCC 13833, CDC80, CDC44, CDC87, CDC47, CDC43, BAA2470, CDC34, CDC39, ATCC 700603 and BAA-2472. The strains tested for P. aeruginosa were CDC263, CDC242, 9721, CDC236, 27853, BAA-2110, CDC233, 15692, CDC234, CDC246 and CDC261. Strains tested for P. mirabilis were CDC155, CDC29, CDC159, CDC59, ATCC 7002, and CDC156. Strains tested for Enterococcus included ATCC 19433, ATCC 29212, ATCC 33186, ATCC 51575, ATCC 51299, and BAA-2128.

[0329] Conclusions: The novel FISH method described in this example detected all strains tested of five different bacterial species that are among the major pathogens causing clinical symptoms in patients with UTI.

[0330] Variations. This example is illustrative of the performance of this novel FISH method and is not limited to the specific details included in the description. Thus, those skilled in the art will easily understand that many variations are possible, including using different probe sequences and nucleic acid structures (PNA, LNA, etc.), alternative assay chemistries (different detergents, chaotropes, fluorophores, buffers, pH, temperature, reaction time, component concentrations), urine concentrations and urine processing procedures. This methodology can obviously be extended to other biological specimens and other bacterial and non-bacterial pathogens.

[0331] Figure 105 shows the average signal (n=3) plotted for 11 E. coli strains 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 indicate 1 standard deviation.

[0332] Figure 18 shows the percentage of input cells detected (determined by plate counting) for each of the 11 E. coli strains. Each bar represents the average of six determinations, in triplicate, from each of two different input cell levels. Percentage cell detection is calculated as [(assay signal - background signal) / input cells] * It was calculated as 100.

[0333] FIG. 19 is a table giving the coveracy results for four additional bacteria.

[0334] FIG. 20 is a table giving the probe sequences used in this example.

[0335] Example 3 Specific detection of target bacteria using rapid isothermal FISH

[0336] Overview. This example demonstrates that the novel isothermal FISH method specifically detects target bacteria without detecting related non-target bacteria, even at very high concentrations. This example shows assay conditions that specifically detect E. coli, but not 16 other bacteria that also cause urinary tract infections (UTIs), have similar rRNA sequences, or are commensals. Experimental method.

[0337] Bacterial cell preparation: Bacterial cultures for the 16 off-target bacteria (listed in Table 1) and E. coli strain ATCC 25922 were grown from single colonies selected from fresh Tryptic Soy Agar plates (TSA, BD catalog 221185), inoculated into Trypticase Soy Broth (TSB, Hardy Diagnostics catalog U65) and grown overnight at 35°C with shaking. 50-80 μL of overnight culture was added into fresh TSB and grown for 1.5-2 hours until the optical density at 600 nm reached 0.15-0.3. Each bacterium was then inoculated at approximately 1 × 10 60 μl into cation-adjusted Mueller Hinton (MHBII, Teknova catalog M5860) at 100 μl. 8 Diluted to cells / mL.

[0338] Selection of bacterial targets to evaluate: Bacterial pathogens to test for specificity were selected for rRNA sequence similarity with the rRNA sequences of the target bacteria, or because they are pathogens commonly found in urinary tract infections (disease targets), and therefore cross-reactivity to these organisms would be most problematic. The table in Figure 21 shows the bacterial species and strains tested.

[0339] Preparation of FISH probes: A DNA probe set for E. coli was heated in a water bath between 80-85°C for 10 minutes and then placed on ice to reduce aggregation. The DNA probe set is shown in the table in Figure 22. The set included species-specific DNA oligonucleotides labeled with a fluorescent dye (Alexa647N, Thermo Fischer) and helper oligonucleotides that bind adjacent to or near the specific probes and are designed to disrupt the local secondary structure of the ribosomal subunits, allowing for higher hybridization efficiency of the labeled specific probes to the target rRNA.

[0340] Preparation of magnetic particles: Polyaspartic acid conjugated magnetic particles (Fluidmag-PAA, Chemicell, catalog 4108), used for non-specific capture of bacterial cells, were diluted to 2.75 × 10 in 50 mM EPPS buffer, pH 8.2. 12 Fluorescent magnetic microspheres containing green dye (Dragon Green Fluorescent Microspheres, BANGS Laboratories, catalog MEDG001) were diluted 1:20 to 3 × 10 6 The magnetic particles were added to the suspension at a final concentration of 10 ...

[0341] Labeling of bacterial cells: A 100 μL labeling reaction was prepared by mixing diluted cells, isothermal hybridization buffer (0.9× MHBII (Teknova catalog M5860), 3× SSC (0.45 M NaCl, 0.045 M sodium citrate, Sigma catalog S6639), 0.77% w / v CHAPSO (Sigma catalog C3649), 0.72% w / v SB3-12 (Sigma catalog D0431), 0.13 M guanidine thiocyanate (Sigma catalog G9277), 0.18% w / v cetrimide (Sigma catalog M7365)), species-specific Alexa647N-labeled probes targeted to 16S or 23S bacterial rRNA (Integrated DNA Technologies, IDT), helper probes to promote efficient hybridization (IDT) and 30 μL of pooled human urine (Innovative A 10 μL magnetic particle preparation was prepared by combining 10 μL of magnetic particle preparation (Thermo Fisher Scientific, Cat. IRHUURE500ML). The specific probe sets tested are shown in the table in FIG. 22. Urine was first processed through a Zeba 7K MWCO spin column (Thermo Fisher, Cat. 89893 or 89892 depending on urine volume) according to the manufacturer's instructions. 10 μL of magnetic particle preparation was then added to this mixture. The final reaction mixture was transferred to a microtiter plate containing 50 μL (pre-dried) per well of "dye cushion" (50 mM TRIS pH 7.5 (Sigma Cat. T1075), 7.5% v / v Optiprep (Sigma Cat. D1556), 50 mg / mL Direct Black 19 (Orient Cat. 191L)) and incubated at 35° C. for 30 minutes to allow simultaneous rehydration of the "dye cushion", labeling of the bacterial cells, and binding of the magnetic particles to the bacterial cell surface. Each bacterium was added at 1×10 per reaction. 6A final concentration of cells was tested, which is approximately 3000 times higher than the determined detection limit for E. coli. After incubation, the microtiter plate was placed on a magnetic field (Dexter magnetic technologies, catalog 54170260) for 4 minutes to bring the magnetic particles, the fraction containing the labeled cells, into close proximity with the imaging surface at the bottom of the wells via a "dye cushion."

[0342] Imaging of Labeled Cells: The MultiPath™ Laboratory Imaging System is a custom instrument and software capable of automatically capturing image data from selected wells of a microtiter plate. It uses a high-precision linear stage from Prior Scientific (Rockland, MA) to position each well on a fluorescence-based image acquisition subsystem. The instrument is capable of imaging in four separate color channels and uses an objective lens, illumination LEDs, a fluorescence filter set, and a camera. The objective lens has a field of view designed to capture an image of the entire microtiter plate well. The illumination module light source consists of two high-power LEDs per color channel. A series of fluorescent image frames are captured by the camera using a 3.1MP Sony IMX265 monochrome sensor with 12 bits per pixel quantization. A final image for each well is then formed by summing multiple frames. 16 frames were captured with a 100 ms exposure using a 635 / 25 nm excitation and 680 / 40 nm emission filter. The focal particle is imaged with 470 / 40 nm excitation and 520 / 40 nm excitation filters, capturing 2 frames with a 20 ms exposure.

[0343] Data analysis: For each bacterium, the number of fluorescent objects was determined (assay signal). Bacteria were considered cross-reactive if a signal was detected within 3 standard deviations of the signal in the blank (no cells added). result.

[0344] Figures 23 and 24 show that the rapid novel FISH method detects only E. coli and not 16 other different challenge bacteria. Figures 1 and 2 each show the detection of E. coli at very high concentrations (1 x 10 per reaction). 6 The results show that eight clinically relevant challenge bacteria (16 cells) are not detected under the same assay conditions that yield a high assay signal for the targeted E. coli bacteria. The two bars represent two different probe sets designed to be specific for E. coli (see table in Figure 22). The assay signal for each of the 16 challenge bacteria was less than the no cells control plus three standard deviations (125).

[0345] Conclusion: The novel rapid FISH method described in this example, by design, specifically detects E. coli but not 16 clinically relevant potentially cross-reactive bacteria, demonstrating that this method has high specificity for the identification of target UTI pathogens that are of great importance for the clinical treatment of infection.

[0346] Variations. This example is illustrative of the performance of this novel FISH method and is not limited to the specific details included in the description. Thus, those skilled in the art will easily understand that many variations are possible, including using different probe sequences and nucleic acid structures (PNA, LNA, etc.), alternative assay chemistries (different detergents, chaotropes, fluorophores, buffers, pH, temperature, reaction time, component concentrations), urine concentrations and urine processing procedures. This methodology can obviously be extended to other biological specimens and other bacterial and non-bacterial pathogens. Assays have also been designed that demonstrate high specificity for K. pneumoniae, K. oxytoca, P. aeruginosa, P. mirabilis and E. faecalis.

[0347] FIG. 21 is a table showing the challenge bacteria to test the specificity of E. coli detection.

[0348] FIG. 22 is a table showing the probe sequences used in this example.

[0349] FIG. 23 shows the specific detection of E. coli and the absence of detection of the eight challenge bacteria.

[0350] FIG. 24 shows the specific detection of E. coli and the absence of detection of eight additional challenge bacteria.

[0351] Example 4 A multiplexed FISH method for simultaneous identification of four distinct microorganisms.

[0352] Overview: This example demonstrates the use of the present invention to simultaneously detect E. coli, K. pneumoniae, P. aeruginosa, and K. oxytoca in a single reaction using fluorescently labeled probes specific for the rRNA of each bacterium. Each pathogen was specifically detected in the mixture through the use of four separate fluorophores - one for each bacterial species - with distinct excitation / emission spectral characteristics.

[0353] Experimental Methods. Bacterial Cell Growth: Bacterial cultures for Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC 13883, Pseudomonas aeruginosa ATCC 27853, and Klebsiella oxytoca ATCC 8724 were obtained by inoculating Trypticase Soy Broth (TSB, Hardy Diagnostics catalog U65) with 3-5 colonies from a fresh Tryptic Soy Agar plate (TSA, BD catalog 221185) and growing at 37°C for 1.5-3 hours to achieve log phase growth. Each culture was then inoculated into cation-adjusted Mueller-Hinton Broth (MHBII, Teknova, catalog M5860) at approximately 1.0 x 10 8 Dilutions were made to give an optical density at 600 nm of 0.15 colony forming units (CFU) / mL.

[0354] 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:

[0355] Labeling of bacterial cells: 100 μL labeling reactions were prepared by combining diluted cells of all four bacteria, isothermal hybridization buffer (0.9× MHBII, 3× SSC (1.5 M NaCl, 0.15 M sodium citrate, Sigma, catalog S6639), 0.77% w / v CHAPSO (Sigma catalog C3649), 0.72% w / v SB3-12 (Sigma catalog D0431), 0.13 M guanidine thiocyanate (Sigma catalog G9277), 0.18% w / v cetrimide (Sigma catalog M7365)), species-specific DNA probes targeted to 16S or 23S bacterial rRNA (Integrated DNA Technologies, IDT), helper probes (IDT) to promote efficient hybridization, and 30 μL of pooled human urine (Innovative Research, catalog IRHUURE500ML). 10 μL of the magnetic particle preparation was then added to the mixture. The probe sequences and the locations of their dye modifications are shown in the table in FIG.

[0356] 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).

[0357] 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.

[0358] FIG. 25 shows a portion of a complete acquired image that detected fluorescence in each of the four color channels, each specific to one of the four input bacteria. Each spot corresponds to a single cell or group of cells. An algorithm is used to identify meaningful objects distinct from artifacts (e.g., debris) and count them as cells. As can be seen in the inset for each bacterium, a similar number of cells was detected as expected, since the input cell concentrations were roughly the same. When overlaid, the spots do not correspond, indicating that with the four different bacterial targets, different objects were observed in each channel, as expected.

[0359] 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:

[0360] 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.

[0361] Figure 25 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.

[0362] FIG. 26 is a table of the probe sequences used in this Example 4.

[0363] Example 6 Automated Rapid AST of E. coli in Clinical Urine Specimens in On-Device Cartridges

[0364] 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:

[0365] 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.

[0366] 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.

[0367] Preparation of magnetic particles: Polyaspartic acid-conjugated magnetic particles (Fluidmag-PAA, Chemicell, catalog 4108) used for nonspecific capture of bacterial cells were dissolved in 50 mM EPPS buffer, pH 8.2 at 2.75 × 10 per mL. 12 Fluorescent magnetic microspheres containing a green dye (Dragon Green Fluorescent Microspheres, BANGS Laboratories, catalog MEDG001) were diluted 1:20 to 3 × 10 particles per mL. 6 The magnetic particles were added to the suspension at a final concentration of 10 ...

[0368] 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.

[0369] 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.

[0370] 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.

[0371] 4 hour growth: While cells were being quantified at time 0, 32.4 μL of processed clinical urine and 75.6 μL of 1.43X MHB II (Teknova, Cat# M5860) were added to each well of the antibiotic plate (which already contained 12 μL of antibiotic). Samples were grown for 4 hours at 35° C. in a standard incubator.

[0372] 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.

[0373] Comparison Methods: Results for the MulitPath™ assay were compared to broth microdilution (BMD) performed according to CLSI method M07-Ed13E 2018.

[0374] 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.

[0375] Figures 27 to 30 show three examples from our larger dataset that demonstrate how this method can be used to obtain MICs for the urine of three individuals that match the gold standard broth microdilution method.

[0376] Figure 27 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.

[0377] Figure 28 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 growth fold threshold.

[0378] Figure 29 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.

[0379] Figure 30 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.

[0380] 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.

[0381] 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.

[0382] FIG. 27 shows BIUR0017 containing nitrofurantoin.

[0383] FIG. 28 shows BIUR047 containing cefazolin.

[0384] FIG. 29 shows BIUR057 containing ciprofloxacin.

[0385] FIG. 30 shows BIUR052 containing trimethoprim / sulfamethoxazole.

[0386] FIG. 31 is a table of the probe sequences used in this Example 6.

[0387] Example 7 Rapid and accurate antimicrobial susceptibility testing for bacteria in urine samples

[0388] 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.

[0389] 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.

[0390] 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.

[0391] 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 ...

[0392] 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. 35. 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.

[0393] 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.

[0394] 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.

[0395] 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.

[0396] 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.

[0397] Comparison Methods: Results for the MulitPath™ assay were compared to broth microdilution (BMD) performed according to CLSI method M07-Ed13E 2018.

[0398] 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 number of growth folds 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.

[0399] Figure 32 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.

[0400] Figure 33 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.

[0401] The table in Figure 34 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.

[0402] 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.

[0403] 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.

[0404] FIG. 32 is a visual comparison of normal bacteria (left panel) against filamentous fungi (right panel).

[0405] FIG. 33 shows that the MIC obtained by the novel, rapid AST method described in this invention is called at 0.25 μg / mL.

[0406] [Table A-1] [Table A-2]

[0407] FIG. 34 is a table of AST results for all bacteria and antibiotics tested in this example.

[0408] FIG. 35 is a table of the probe sequences used in this Example 7.

[0409] Example 8 Rapid and accurate automated AST results for clinical urine samples without cell purification

[0410] 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.

[0411] 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.

[0412] 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.

[0413] 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

[0414] A few days prior to preparing the cartridges (see FIG. 1 for a diagram of the cartridge), 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 ciprofloxacin (CIP) and nitrofurantoin (NIT) or 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.

[0415] Preparation of AST Cartridges - Hybridization Reagents

[0416] 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 an 8.3 μL volume of beads. Two 8.3 uL beads were placed into each of the eight reagent wells (see FIG. 1 for location on the cartridge). Preparation of AST Cartridges - Magnetic Particles

[0417] 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). 12One 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

[0418] 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

[0419] 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

[0420] 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.

[0421] 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

[0422] 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.

[0423] 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.

[0424] Figure 37 shows the average growth fold of four replicates in two cartridges containing clinical urine sample BIUR0067 that contained an E. coli strain. The graph shows the average growth fold at each of two concentrations of each of ciprofloxacin and nitrofurantoin across four replicates in two different cartridges. Using a growth fold value of 2 for both antibiotics, the MulitPath assay calls both ciprofloxacin (CIP) concentrations as growth and both nitrofurantoin (NIT) concentrations as no growth. Thus, by MulitPath, BIUR0067 is resistant to ciprofloxacin and susceptible to nitrofurantoin. E. coli strains isolated from this urine and tested in CLSI-standard broth microdilution were consistent with these susceptibility / resistance calls.

[0425] Figure 39 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.

[0426] 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.

[0427] 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.

[0428] FIG. 36 shows the Multipath™ UTI-AST cartridge.

[0429] FIG. 37 shows a table of the antibiotic concentrations tested.

[0430] FIG. 38 is a table of the oligonucleotides used in this Example 8.

[0431] Figure 39 shows the results for BIUR0067.

[0432] Figure 40 shows the results for BIUR0084.

[0433] Example 9 Direct rapid AST methods in urine samples are robust to a range of pathogen concentrations

[0434] 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.

[0435] Experimental procedure.

[0436] 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.

[0437] 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.

[0438] 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 ...

[0439] 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.

[0440] 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.

[0441] 4 hour growth: While the cells were being quantified at time 0, 10 μL of each organism inoculum, 10 μL of pooled urine, and 70 μL of 1× MHBII were added to the appropriate wells of an antibiotic plate (already containing 10 μL of antibiotic). Samples were grown for 4 hours at 35° C. in a standard air incubator.

[0442] 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.

[0443] Comparison Methods: Results using the novel AST method described herein were compared to broth microdilution (BMD) performed according to CLSI M07-Ed13E 2018.

[0444] Data analysis and threshold creation: Images captured by the CCD camera were used to estimate the detected cells by an algorithm that takes into account both the number of objects in the field of view and the intensity of those objects. Cell counts based on this detection algorithm were obtained at time 0, as well as at time 4 hours without antibiotic and at time 4 hours with all concentrations of each antibiotic. For each sample inoculum per drug concentration, the growth fold was calculated as the signal in the wells containing antibiotic after growth (time 4) relative to the signal in the urine sample before growth (time 0). Using the growth fold and the observation of growth in the corresponding wells in CLSI-compatible broth microdilution, a logistic regression model was used to create a threshold to determine a growth fold cutoff above where cells are growing in the presence of antibiotic (and thus resistant at that concentration) and below where cells are in the process of dying (and thus sensitive at that concentration). The point where the growth fold number is below the determined threshold is the MIC value generated by the assay. Correspondingly, the results were assigned to a category of sensitive, intermediate, or resistant to each antibiotic. All data were then compared to the CLSI standard BMD. Four hours of growth in the absence of antibiotics is the control condition to confirm that viable bacteria are present in the treated urine samples. result.

[0445] The figures below show how this method is robust to changes in inoculum levels while concordant with the gold standard broth microdilution method.

[0446] Figure 41 compares the results obtained by the new AST method with those of the standard BMD performed at a single concentration for all drugs tested. The third row compared the MICs obtained by the new AST method and the gold standard BMD. All MIC calls were within one two-fold dilution of the CLSI corresponding BMD (essential agreement). The fourth row compared categorical antibiotic susceptibility results (S=susceptible, I=intermediate, R=resistant) based on MICs (categorical agreement). Although concentrations of a subset of Klebsiella give different category calls from the MICs in broth microdilutions, all of these were classified as only minor errors by standard AST methodology.

[0447] Figure 42 shows the MICs (filled circles) obtained by the novel 4-hour method described above for all inoculum levels against E. coli BAA-2469 compared to the standard broth microdilution method (24-hour BMD, dashed line). All MICs determined by the novel method were within essential agreement (shaded areas). Figure 43 shows the raw data for Figure 42.

[0448] Conclusion: The rapid 4-h AST method presented herein is robust to initial cell concentrations over a wide range of target cell concentrations. Robustness to variable inoculum concentrations is important for rapid AST methods because the concentration of target cells is unknown when testing specimens derived directly from the specimen.

[0449] Variations. This example is illustrative of the performance of this novel AST method and is not limited to the specific details contained herein. Those skilled in the art will therefore readily appreciate that many variations are possible, including using different probe sequences and nucleic acid structures (PNA, LNA, etc.), alternative assay chemistries (different detergents, chaotropes, fluorophores, buffers, pH, temperature, reaction time, concentrations of components), urine concentrations and urine processing procedures. This methodology can also obviously be extended to other biological specimens and other bacterial and non-bacterial pathogens for which specific probes can be designed, as well as to other antimicrobial or chemical agents.

[0450] FIG. 41 is a summary of the overall intrinsic and categorical agreement for all organisms, antibiotics and inoculum levels.

[0451] FIG. 42 shows the MIC results for various inoculum levels obtained using the new method described herein compared to the conventional BMD method.

[0452] FIG. 43 summarizes the MIC results obtained for the various inoculum levels.

[0453] FIG. 44 is a table of the probe sequences used in this Example 9.

[0454] Example 10 Rapid antimicrobial susceptibility testing for target pathogens in urine clinical specimens containing multiple bacterial species without cell purification

[0455] Overview. Current methods for antimicrobial susceptibility testing require lengthy culture-based colony purification to ensure a pure population of target pathogen cells free of other microorganisms. Colony purification, the usual method, requires 2-5 days to deliver results. During that time, patients are empirically treated with potent broad-spectrum antibiotics that may not be optimal or even completely ineffective at killing the pathogen causing the infection. Additionally, empirical treatment with broad-spectrum antibiotics leads to the spread of antibiotic resistance.

[0456] Current methods require lengthy cell purification processes to determine which antimicrobial agents inhibit the growth of the target pathogen in microbiological media because these methods use non-specific detection methods such as increased turbidity. When using non-specific measurements of cell replicas, one can only know that the growth seen is due to the target pathogen if only cells of the target pathogen are contained. Current antimicrobial susceptibility testing methods must undergo cell purification because most medical specimens are not sterile. The specimens generally contain microorganisms that make up the human microbiome, the benign, normal population of bacteria that live in the human body.

[0457] In contrast, the method of the present invention can achieve accurate antimicrobial susceptibility test results directly from the specimen without a colony purification step. This method differs from current methods in that it evaluates target pathogen-specific growth in a microbiological medium containing an antimicrobial agent.

[0458] This example demonstrates that the rapid antimicrobial susceptibility testing method accurately determines minimum inhibitory concentrations (MICs) for E. coli strains in artificial samples containing urine matrix (10%) for 15 different culture-negative urine samples. Here, we show that antimicrobial susceptibility testing results using the new method are accurate and not significantly affected by off-target bacteria in urine samples containing high concentrations of other microbial species. Experimental procedure.

[0459] Preparation of antibiotic plates: Prior to beginning the experimental procedure, plates containing five concentrations in a two-fold serial dilution series were prepared by dispensing 10 μL of a concentration ten times higher than the desired concentration. The concentrations chosen to test each antibiotic spanned the breakpoints reported by CLSI for antibiotics against E. coli. In addition to the wells containing the antimicrobial dilution series, four wells containing water were included in the plate to allow for positive and negative controls.

[0460] Culture preparation: Three to five colonies of E. coli BAA-2469 and eight other off-target species (S. aureus ATCC 25923, C. freundii ATCC 43864, A. baumannii ATCC 19606, S. epidermidis ATCC 12228, M. luteus (environmental isolate), C. minutissmum ATCC 23348-BAA 949, K. pneumoniae CDC 0043, and K. pneumoniae CDC 0141) were each separately inoculated into 5 mL of Tryptic Soy Broth (TSB, Hardy Diagnostics catalog U65) and incubated at 35°C for 1-2 hours with shaking. The optical density was measured by spectrophotometer and the organisms were diluted in Mueller-Hinton Broth (MHBII, Teknova catalog M5860) adjusted with 1X cations. E. coli was cultured at approximately 5 × 10 6 CFU / mL (final assay concentration is 5 × 10 5 CFU / m), while other off-target species were diluted for various inocula (1 × 10 5 From 5×10 8 CFU / mL).

[0461] 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 ...

[0462] Labeling of bacterial cells at AST time 0: The assay signal before the onset of bacterial growth (time 0 or T0) was determined for each species of E. coli in the presence or absence of antibiotics. 10 μL of each sample was added to 80 μL of hybridization buffer (3X SSC (0.45 M NaCl, 0.045 M sodium citrate) (Sigma, Cat. No. S6639), 1% CHAPS (Sigma Cat. No. C3023), 1% SB3-12 (Sigma Cat. No. 08001), 1X Mueller-Hinton Broth adjusted with cations (MHBII), E. coli-specific DNA oligonucleotide FISH probes and unlabeled DNA helper probes). The probe sequences are shown in the table in Figure 50. A final concentration of 9.1% urine was obtained by adding 10 μL of pooled urine (collected and filtered in-house) directly to the mixture. 10 μL of the magnetic particle mixture prepared as described above was added directly to this mixture. Here, 100 μL of sample containing hybridization mixture, urine, and magnetic particles were transferred to a microtiter plate containing 50 μL of "dye cushion" (50 mM TRIS pH 7.5 (Teknova, Cat. T5075), 7.5% v / v Optiprep (Sigma, Cat. D1556), 5 mg / mL Direct Black-19 (Orient, Cat. No. 3222) per well (previously dried) and incubated at 35° C. for 30 minutes. After incubation, the microtiter plate was placed on a magnetic field (Dexter magnetic technologies, Cat. 54170260) for 4 minutes, allowing the fraction containing the magnetic particles, labeled cells, to migrate through the "dye cushion" near the imaging surface at the bottom of the well.

[0463] 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.

[0464] Four hours of growth: E. coli BAA 2469 was tested for their susceptibility to three antimicrobial agents: ciprofloxacin (CIP), levofloxacin (LVX), and nitrofurantoin (NIT) in the presence of Staphylococcus epidermidis, Micrococcus luteus, Corynebacterium minutissimum, Staphylococcus aureus, Acinetobacter baumannii, and Citrobacter freundii. E. coli BAA 2469 was tested against five antimicrobial agents: cefazolin (CFZ), ciprofloxacin (CIP), levofloxacin (LVX), nitrofurantoin (NIT), and trimethoprim-sulfamethoxazole (TMP / SXT) in the presence of Klebsiella pneumoniae. Antibiotic plates containing these antimicrobial agents were prepared according to the method described above. While the cells were quantified at time 0, either E. coli species (5 × 10 6 CFU / mL) 10 μL, off-target species (1 × 10 5 From 5×10 8 10 μL of 100 μL of 1000 CFU / mL, 10 μL of pooled urine, and 60 μL of MHB II (Teknova, Cat# M5860) were added to each well of the antibiotic plate already containing 10 μL of antibiotic. Samples were grown for 4 hours at 35° C. in a static air incubator.

[0465] Labeling of bacterial cells at time 4 hours of growth in AST: After incubating samples for 4 hours (T4) in the presence and absence of antibiotics, cells were labeled and quantified to determine how much growth, if any, had occurred. 10 μL (10%) of the incubated sample-antibiotic plate was transferred to a microtiter plate containing a dried "dye cushion" and combined with 100 μL of the mixture of hybridization buffer, FISH probes, helper probes, magnetic particles, and focusing particles as described above for the time 0 assay.

[0466] Comparison Method: Results from the novel assay method described herein were compared to broth microdilution (BMD) performed according to M07-Ed13E 2018.

[0467] Data analysis and threshold creation: Images captured by the CCD camera were used to estimate the cells detected by an algorithm that takes into account both the number of objects in the field of view and the intensity of those objects. Cell counts based on this detection algorithm were obtained at time 0, as well as at time 4 hours without antibiotic and at time 4 hours with all concentrations of each antibiotic. For each bacterial sample per drug concentration, the growth fold was calculated as the signal in the well containing antibiotic after growth (time 4) relative to the signal in the urine sample before growth (time 0). Using the growth fold and the observation of growth in the corresponding well in CLSI-compatible broth microdilution, a logistic regression model was used to create a threshold to determine a growth fold cutoff above where cells are growing in the presence of antibiotic (and thus resistant at that concentration) and below where cells are in the process of dying (and thus sensitive at that concentration). The point at which the growth fold number is below the determined threshold is the MIC value generated by the assay. Correspondingly, the results were assigned to categories of susceptible, intermediate, or resistant to each antibiotic. result.

[0468] The data presented demonstrates that the 4 hour AST method is robust to non-sterile samples, whereas the CLSI BMD method in the presence of excess bacteria is not robust.

[0469] Figure 47 shows data for E. coli BAA-2469 with increasing concentrations of S. aureus ATCC 25923 up to 100-fold excess in the presence of nitrofurantoin. The MIC of E. coli in the CLSI-like broth microdilution method is affected by the addition of S. aureus strains (marked with an X in the figure), where the MIC increases from 8 in the absence of S. aureus to 32 with a 100-fold excess of S. aureus. In contrast, the novel 4-hour AST assay described in this invention (MultiPath, circles) had the same MIC (8) (dashed line) regardless of the amount of S. aureus cells.

[0470] Figures 47-49 show the raw MIC values ​​determined using this novel method (MultiPath) compared to CLSI broth microdilution with only E. coli BAA-2469 present. The table in Figure 46 shows the overall essential agreement for E. coli in the presence of increasing off-target bacteria. Only a single condition - nitrofurantoin with 1e7 Citrobacter freundii - resulted in a lack of essential agreement, but this did not change the categorical susceptible / intermediate / resistant determination, which had 100% agreement across all antibiotics and all off-target bacteria.

[0471] Conclusion: This example demonstrates that using the present invention for antimicrobial susceptibility testing, no cell purification is required to achieve accurate antimicrobial susceptibility test results for the target pathogen, even in samples containing large numbers of other microorganisms of other species.

[0472] Variations: This example is illustrative of the performance of this novel FISH method and is not limited to the specific details contained herein. Those skilled in the art will therefore readily understand that many variations are possible, including using different probe sequences and nucleic acid structures (PNA, LNA, etc.), alternative assay chemistries (different detergents, chaotropes, fluorophores, buffers, pH, temperature, reaction time, concentrations of components), urine concentrations and urine processing procedures. This methodology can also clearly be extended to other biological specimens and other bacterial and non-bacterial pathogens.

[0473] FIG. 45 shows that the MICs for E. coli are consistent with the method above for various inoculations of S. aureus, while the MICs for BMD increase with increasing S. aureus.

[0474] FIG. 46 shows a summary of the concordance for E. coli with various inoculation levels of off-target microorganisms against standard BMD.

[0475] FIG. 47 shows the concordance of E. coli with various inoculum levels of standard BMD of off-target microorganisms (S. aureus, Staphylococcus epidermidis, and Citrobacter freundii).

[0476] FIG. 48 shows the concordance of E. coli with various inoculum levels of standard BMD of off-target microorganisms (Micrococcus luteus, Acinetobacter baumannii, Corynebacterium minutissimum).

[0477] FIG. 49 shows the concordance of E. coli with various inoculum levels of the standard BMD of an off-target microorganism (K. pneumoniae).

[0478] FIG. 50 is a table of the probe sequences used in this Example 10.

[0479] Example 11 Rapid antimicrobial susceptibility testing is accurate for lactam antibiotics in the presence of beta-lactamase-expressing bacteria

[0480] Overview. Current methods for antimicrobial susceptibility testing require lengthy culture-based colony purification to ensure a pure population of target pathogen cells free of other microorganisms. Colony purification, the usual method, requires 2-5 days to deliver results. During that time, patients are empirically treated with potent broad-spectrum antibiotics that may not be optimal or even completely ineffective at killing the pathogen causing the infection. Additionally, empirical treatment with broad-spectrum antibiotics leads to the spread of antibiotic resistance.

[0481] One of the reasons current methods require lengthy cell purification processes to determine which antimicrobial agents inhibit the growth of a target pathogen in a microbiological medium is because they use non-specific detection methods such as increased turbidity. When using non-specific measurements of cell replication, one can only know that the growth seen is due to the target pathogen if only cells of the target pathogen are contained.

[0482] In contrast, the method of the present invention can achieve accurate antimicrobial susceptibility test results directly from specimens without a colony purification step. This method differs from current methods in that it evaluates target pathogen-specific growth in microbiological media containing antimicrobial agents. In another example, the inventors demonstrate that the method of the present invention is accurate in the presence of large numbers of cells from off-target species.

[0483] In this example, we address another challenge that may arise from performing antimicrobial susceptibility testing on target pathogens in the presence of off-target species. Here, we demonstrate that the method of the present invention achieves accurate antimicrobial susceptibility testing results on target pathogens in artificial urine specimens that contain a large number of off-target species that create enzymes known to degrade the antimicrobial agent being tested. In theory, this could change the concentration of the antimicrobial agent significantly enough to alter the results of the antimicrobial susceptibility test.

[0484] In this example, we demonstrate that rapid antimicrobial susceptibility testing achieves accurate antimicrobial susceptibility test results for two carbapenem antibiotics (meropenem and imipenem) in the presence of numerous off-target pathogens that produce enzymes that degrade this type of antimicrobial.

[0485] Experimental Procedures. Preparation of antibiotic plates: Antibiotic plates were prepared as described in the example of the effect of non-sterile samples on target MICs.

[0486] Preparation of cultures: Three to five colonies of E. coli ATCC 25922, a bacterial strain susceptible to most antibiotics, and K. pneumoniae CDC 0141, a strain that expresses the beta-lactamase OXA-181, among many other resistance genes, were separately inoculated into 5 mL of Tryptic Soy Broth (TSB, Hardy Diagnostics catalog U65) and incubated at 35°C for 1-2 hours with shaking. Optical density was measured by spectrophotometer, and organisms were diluted into Mueller-Hinton Broth (MHBII, Teknova catalog M5860) adjusted with 1X cation. E. coli was diluted to 5×10 5 CFU / mL (CLSI standard concentration), while K. pneumoniae was diluted for various inoculations (1 × 10 6 From 5×10 8 CFU / mL).

[0487] Magnetic particle preparation: 2-hydroxypropyltrimethylammonium chloride coated silica magnetic particles (SiMag-Q, Chemicell, catalog 1206-5) were dissolved in 50 mM EPPS buffer, pH 8.2 at 3.75 × 10 per mL. 6 Fluorescent magnetic microspheres containing a green dye (Dragon Green Fluorescent Microspheres, BANGS Laboratories, catalog MEDG001) were diluted 1:20 to 3 × 10 particles per mL. 6 The magnetic particles were added to the suspension at a final concentration of 10 ...

[0488] 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 a well of a microtiter plate containing dehydrated hybridization buffer (3X SSC (0.45 M NaCl, 0.045 M Na Citrate) buffer (Sigma, Catalog No. S6639), 0.18% Cetrimide (Sigma, Catalog No. H9151), 0.77% CHAPSO (Sigma Catalog No. C3649), 0.72% SB3-12 (Sigma Catalog No. D0431) 0.13 M Guanidine Thiocyanate (Sigma, Catalog No. G9277)). 10 μL of the prepared magnetic particle mixture was then added to the well. 100 μL of this reaction mixture was transferred to a microtiter plate containing 50 μL of "dye cushion" (50 mM TRIS pH 7.5 (Sigma Cat. T1075), 7.5% v / v Optiprep (Sigma Cat. D1556), 50 mg / mL Direct Black 19 (Orient Cat. 191L)) per well (pre-dried) and incubated for 30 minutes at 35° C. After incubation, the microtiter plate was placed on a magnetic field (Dexter magnetic technologies, Cat. 54170260) for 4 minutes, allowing the fraction containing the magnetic particles, the labeled cells, to migrate through the "dye cushion" near the imaging surface at the bottom of the well.

[0489] 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 obj...

Claims

1. 1. A cartridge configured to perform one of a variety of tests on a specimen, including detection and quantification of individual cellular and molecular targets and antimicrobial susceptibility testing, the cartridge comprising: A specimen chamber; a plurality of dividing wells which can be selected to be either in fluid communication or not in fluid communication with said analyte chamber; a plurality of reagent wells in selectable fluid communication with the divided wells, each reagent well containing at least one reagent; a plurality of imaging wells, each imaging well in fluid communication with a corresponding one of the plurality of reagent wells; a slidable valve movable to a plurality of positions operable from outside the cartridge to open and close channels of fluid communication between the sample chamber and the plurality of divided wells and between the plurality of divided wells and the plurality of reagent wells, the slidable valve including a channel providing direct fluid communication between the sample chamber and at least one divided well of the plurality of divided wells when the slidable valve is in a first position of the plurality of positions and providing a connection from the at least one divided well to a corresponding reagent well when the slidable valve is in a second position of the plurality of positions; and a pneumatic port configured to interface with a fluidics module to manipulate pressure within the cartridge to move analytes within the cartridge; wherein pressure or vacuum applied to the pneumatic port, in combination with a position of the slidable valve, is configured to control dispensing of the sample and the at least one reagent.

2. The cartridge of claim 1 , wherein the at least one reagent comprises a magnetic particle and a target-specific or non-specific label.

3. The cartridge of claim 2 , wherein the magnetic particles are not target specific.

4. The cartridge of claim 3 , wherein the magnetic particles are capable of non-specifically binding to a bacterial cell surface.

5. The cartridge of claim 2 , wherein each of the target-specific or non-specific labels comprises a fluorescently labeled oligonucleotide complementary to a segment of the ribosomal RNA of a particular bacterial target.

6. The cartridge of claim 1 , wherein each imaging well comprises a detection surface and a dye cushion.

7. 7. The cartridge of claim 6, wherein the dye cushion comprises a density agent and a dye adjacent to the detection surface, the at least one reagent comprises magnetic particles, and the magnetic particles and dye cushion are configured such that upon application of a magnetic field across the dye cushion, the magnetic particles and targets bound thereto are attracted through the dye cushion to the detection surface.

8. The cartridge of claim 2 , wherein the magnetic particles and the target-specific or non-specific labels are provided as lyophilized beads that are rehydrated and dissolved upon delivery of the analyte into the reagent well.

9. The cartridge of claim 2 , wherein the target-specific or non-specific label is taxon-specific.

10. The cartridge of claim 2 , wherein the magnetic particles are capable of non-specifically binding to microbial cells.

11. The cartridge of claim 2 , wherein the magnetic particles are configured to bind to specific features or molecules on the surface of bacterial cells.

12. a diluent reservoir having a diluent therein; and a button configured, when pressed, to dispense the diluent from the diluent reservoir to mix with the specimen; The cartridge of claim 1 further comprising:

13. The slidable valve is retaining said specimens in said divided wells during incubation; Delivering the analyte from the partition well to the reagent well for labeling and magnetic tagging. The cartridge of claim 1 , configured to be arranged to

14. The cartridge of claim 1 , wherein the slidable valve is in the second position and each divided well is in fluid communication with a corresponding reagent well.

15. The cartridge of claim 1 , further comprising an identifier comprising patient, test application specific and factory information associated with the cartridge.

16. The cartridge of claim 1, further comprising focusing particles therein that enable the optical system of the analyzer to focus onto a focal plane adjacent to the detection surface where the labeled, magnetically tagged target cells or molecules are deposited by magnetic force.

17. 10. The cartridge of claim 1, wherein the cartridge has an asymmetric footprint to allow insertion of the cartridge into an analyzer tray in only one orientation to avoid clogging or other errors.

18. The cartridge of claim 1 , wherein at least one of the divided wells contains one or more antimicrobial agents.

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