Systems, methods, and devices for antimicrobial susceptibility testing

Abstract

Described herein are systems, methods, and devices for performing antimicrobial susceptibility testing (AST) directly from a blood sample. The system for concentrating a sample includes a housing configured to receive a sample container, a sample preparation cartridge, and an AST cartridge, and processes and tests the sample using one or more centrifuges, a pipetting system, a controller, an AST subsystem, a magnet station, a microscope, and a heater disposed within the housing. The system is configured to transfer the sample from the blood sample container to a processing tube in the sample preparation cartridge using a septum and needle-based pipetting system. After sample transfer, the system performs steps to separate, concentrate, and enrich pathogens in the sample for rapid detection. Aliquots of the enriched sample are then dispensed into antimicrobial-containing reaction wells in the AST cartridge, images of each reaction well are acquired after incubation, and susceptibility of the pathogens to the antimicrobial is determined based on analysis of the acquired images.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 299,555, filed January 14, 2022, the entire contents of which are incorporated herein by reference. This application is related to co-pending U.S. Patent Application No. ___, filed January 13, 2023 (Attorney Docket No. 4518.003PC01), entitled "SYSTEMS, METHODS, AND APPARATUS FOR PATHOGEN IDENTIFICATION," by Bru Gibert et al., and PCT Application No. PCT / US2022 / 039290, filed August 3, 2022, the disclosures of which are incorporated herein by reference in their entireties.

[0002] Embodiments of the present disclosure relate to systems, methods, and devices for performing antimicrobial susceptibility testing (AST) from whole blood or other samples to determine patient treatment. [Background technology]

[0003] Sepsis is defined as life-threatening organ dysfunction caused by an uncontrolled host response to infection. Patients with or developing sepsis may begin with a localized infection, such as pneumonia, to which the patient's immune system overreacts, causing inflammation throughout the body. If left untreated, inflammation can eventually lead to organ failure and patient death. Sepsis causes 11 million deaths each year, many of which are preventable with early diagnosis, appropriate clinical management, and treatment.

[0004] The diagnosis of sepsis is performed by laboratory testing of cultured blood samples to identify infection. Current testing can take several days to produce test results due to the time required to collect and process blood cultures for the bacteria causing sepsis to determine their susceptibility to antimicrobial agents that will ultimately be effective in treating them. However, for patients in hospitals, detection of sepsis is a race against time, as sepsis can lead to septic shock and death within hours if not properly identified and treated in time. Furthermore, blood culture testing is time-consuming and does not always produce reliable results in detecting bacteria and fungi in patients suspected of sepsis, especially for patients who have already been treated with antibiotics. The probability of a positive result in a patient's blood culture is low, and patients may still have sepsis even without identification of a positive result.

[0005] Traditional and automated methods for performing antimicrobial susceptibility testing require the preparation of a “pure culture” of an isolated bacterium and long incubation times (e.g., 6–24 hours) for bacterial growth. These traditional systems are limited in that they are growth-based, time-consuming, expensive, manual, and non-integrated. Without a remedy, patients may continue to suffer and often worsen while physicians treat them with empirical antibiotics until they receive more actionable information from the laboratory about the causative agents (e.g., pathogens) causing the infection, and about antimicrobials to treat highly resistant pathogens in patients who have an infection. Summary of the Invention

[0006] Embodiments of the present disclosure provide cost-effective solutions for improved diagnostic methods, systems, and devices for antimicrobial susceptibility testing with the goal of providing appropriate treatment to patients to improve their prognosis.

[0007] Described herein are systems, methods, and devices for performing antimicrobial susceptibility testing (AST) directly from blood samples or other samples such as urine, sterile body fluids, etc. In embodiments presented herein, AST systems, analyzers, AST cartridges, sample preparation cartridges, and processing tubes are provided to rapidly perform AST from samples without blood culture while maintaining the specimen quality of the sample. In some embodiments, single cell microscopy is utilized to identify phenotypical susceptibility of pathogens, which may lead to a diagnostic pathway for rapid and effective antimicrobial treatment. In some embodiments, the systems, methods, and devices for AST described herein may be used to treat patients with sepsis and / or other underlying diseases.

[0008] In one embodiment, an example of a method for performing susceptibility testing is described, the method includes receiving, by an analytical device, a sample preparation cartridge and a sample container, the sample container containing a sample including a pathogen, the method also includes placing a first needle from the sample preparation cartridge into a pipettor system in the analytical device, inserting the first needle into the sample container using the pipettor system, transferring the sample from the sample container through the first needle to a processing tube in the sample preparation cartridge, concentrating and enriching the pathogen of the sample in the processing tube using the analytical device to obtain an enriched sample in the processing tube, and dispensing a plurality of aliquots of the enriched sample into a plurality of reaction wells in an antimicrobial susceptibility testing (AST) cartridge in the analytical device, each aliquot corresponding to a respective reaction well, each reaction well containing a predetermined concentration of an antimicrobial. The method further includes the steps of incubating the aliquots in the reaction wells of the AST cartridge for a predetermined period of time to allow a reaction to occur between the pathogens in each reaction well and the antimicrobial agent in each reaction well; acquiring images of each reaction well in the AST cartridge using a microscope in the analyzer; and analyzing the images with a processor coupled to the microscope in the analyzer to determine the susceptibility of the pathogens in each reaction well to the antimicrobial agent in each reaction well.

[0009] In another embodiment, an exemplary antimicrobial susceptibility testing (AST) cartridge is described. The AST cartridge includes a base including a plurality of reaction wells, each reaction well including a bottom wall, the bottom wall being optically transparent. The AST cartridge further includes a septum disposed on the base that seals each reaction well in the plurality of reaction wells, and a cover disposed on the septum. Each reaction well in the plurality of reaction wells contains a predetermined concentration of an antimicrobial agent for reacting with a respective aliquot of an enriched sample containing a pathogen, the antimicrobial agent being disposed in each reaction well.

[0010] In another embodiment, an example of a system for enriching a sample is described. The system includes a housing configured to receive a sample container containing a sample including a pathogen, a pipetter system disposed within the housing, one or more centrifuges disposed within the housing, and a controller. The controller is configured to transfer the sample from the sample container to a processing tube using the pipetter system, centrifuge the processing tube using the one or more centrifuges to concentrate pathogens in the sample, remove fluid from the processing tube using the pipetter system to leave the concentrated pathogens in the processing tube, add growth media to the concentrated pathogens in the processing tube using the pipetter system, grow the concentrated pathogens in the processing tube for a predetermined period of time, and wash the concentrated pathogens after the predetermined period to obtain an enriched sample in the processing tube.

[0011] In another embodiment, an example of a system for analyzing a sample is described. The system includes a housing configured to receive a processing tube and an antimicrobial susceptibility testing (AST) cartridge. The AST cartridge includes a plurality of reaction wells, each reaction well includes a bottom wall, and the bottom wall is optically transparent. The system further includes a heater disposed within the housing, a pipettor system disposed within the housing, a microscope disposed within the housing, and a controller. The controller is configured to use the pipettor system to dispense a plurality of aliquots of an enriched sample including a pathogen from the processing tube into a plurality of reaction wells in the AST cartridge, each aliquot corresponding to a respective reaction well, and each reaction well includes a predetermined concentration of an antimicrobial agent. The controller is further configured to use the heater to incubate the aliquots in the reaction wells of the AST cartridge for a predetermined period of time to allow a reaction to occur between the pathogen in each reaction well and the antimicrobial agent, and to use the microscope to acquire one or more images of the bottom wall of each reaction well in the AST cartridge, and to determine the susceptibility of the pathogen in each reaction well to the antimicrobial agent by analyzing the one or more images by a processor coupled to the microscope.

[0012] In another embodiment, an example method for manufacturing an AST cartridge is described, the method includes manufacturing a cover with a plurality of openings, and overmolding a septum onto the cover, where a first side of the septum extends across the plurality of openings, the method also includes manufacturing a base with a plurality of reaction wells, and attaching the base to a second side of the septum, where the second side of the septum extends across and seals the plurality of reaction wells of the base.

[0013] Further features and advantages, as well as the structure and operation of various embodiments, are described in detail below with reference to the accompanying drawings. It should be noted that the specific embodiments described herein are not intended to be limiting. Such embodiments are presented herein for illustrative purposes only. Further embodiments will be apparent to one of ordinary skill in the relevant art based on the teachings set forth herein. [Brief description of the drawings]

[0014] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the disclosure and, together with the description, further explain the principles of the disclosure and enable those skilled in the art to make and use the disclosure.

[0015] [Figure 1] FIG. 1 shows a diagram of a system for performing antimicrobial susceptibility testing (AST) according to an embodiment of the present disclosure. [Diagram 2] FIG. 2 illustrates an analytical device according to an embodiment of the present disclosure. [Figure 3A] FIG. 3A shows a front view of an analytical device according to an embodiment of the present disclosure. [Figure 3B] FIG. 3B illustrates a top view of an analytical device according to an embodiment of the present disclosure. [Figure 4] FIG. 4 shows a diagram of an analytical device with three open compartments according to an embodiment of the present disclosure. [Diagram 5] FIG. 5 shows a diagram of a sample preparation cartridge according to an embodiment of the present disclosure. [Figure 6A] FIG. 6A shows a processing tube and other components in a sample preparation cartridge according to an embodiment of the present disclosure. [Figure 6B] FIG. 6B shows processing tubes and other components in a linear sample preparation cartridge according to an embodiment of the present disclosure. [Figure 7A] FIG. 7A shows a diagram of a processing tube according to an embodiment of the present disclosure. [Figure 7B] FIG. 7B shows a diagram of a processing tube according to an embodiment of the present disclosure. [Figure 8A] FIG. 8A shows a diagram of a needle configured for insertion into a process tube according to some embodiments of the present disclosure. [Figure 8B] FIG. 8B shows a diagram of a needle configured for insertion into a process tube according to some embodiments of the present disclosure. [Figure 8C] FIG. 8C shows a diagram of a needle configured for insertion into a processing tube according to some embodiments of the present disclosure. [Figure 9A] FIG. 9A shows a diagram of a high volume needle according to an embodiment of the present disclosure. [Figure 9B] FIG. 9B shows a diagram of a low volume needle according to an embodiment of the present disclosure. [Figure 10] FIG. 10 shows a diagram of an example high volume needle according to an embodiment of the present disclosure. [Figure 11] FIG. 11 illustrates how a low volume needle interfaces with a sample preparation cartridge according to an embodiment of the present disclosure. [Figure 12A] FIG. 12A shows a diagram of an AST cartridge according to an embodiment of the present disclosure. [Figure 12B] FIG. 12B shows a diagram of an AST cartridge according to an embodiment of the present disclosure. [Figure 13] FIG. 13 illustrates a low volume needle being inserted into an AST cartridge according to an embodiment of the present disclosure. [Figure 14A] FIG. 14A shows a diagram of an example centrifuge for use in an analytical device according to an embodiment of the present disclosure. [Figure 14B] FIG. 14B shows a diagram of an example centrifuge for use in an analytical device according to an embodiment of the present disclosure. [Figure 15A] FIG. 15A shows a diagram of one example of an enrichment subsystem for use in an analysis device according to an embodiment of the present disclosure. [Figure 15B] FIG. 15B shows a diagram of an example of an enrichment subsystem for use in an analysis device according to an embodiment of the present disclosure. [Figure 15C] FIG. 15C shows a diagram of an example of an enrichment subsystem for use in an analyzer according to an embodiment of the present disclosure. [Figure 15D] FIG. 15D shows a diagram of one example of an enrichment subsystem for use in an analysis device according to an embodiment of the present disclosure. [Figure 16A] FIG. 16A shows a diagram of an example of a mechanical device used in an analysis device according to an embodiment of the present disclosure. [Figure 16B] FIG. 16B shows a diagram of an example of a mechanical device used in an analysis device according to an embodiment of the present disclosure. [Figure 16C] FIG. 16C shows a diagram of an example of a mechanical device used in an analysis device according to an embodiment of the present disclosure. [Figure 17A] FIG. 17A shows a diagram of an AST cartridge interfacing with an imaging subsystem in an analyzer according to an embodiment of the present disclosure. [Figure 17B] FIG. 17B shows a diagram of an AST cartridge interfacing with an imaging subsystem in an analyzer according to an embodiment of the present disclosure. [Figure 18] FIG. 18 shows a flow chart diagram of a method for performing AST of a sample according to an embodiment of the present disclosure. [Figure 19] FIG. 19 shows a flow chart diagram of a method for manufacturing or assembling an AST cartridge according to an embodiment of the present disclosure. [Figure 20] FIG. 20 is a block diagram of exemplary components of a computer system in accordance with an embodiment of the present disclosure. [Figure 21A] FIG. 21A shows experimental results from a test according to an embodiment of the present disclosure. [Figure 21B] FIG. 21B shows experimental results from testing according to an embodiment of the present disclosure. [Figure 22A] FIG. 22A shows experimental results from testing according to an embodiment of the present disclosure. [Figure 22B] FIG. 22B shows experimental results from testing according to an embodiment of the present disclosure. [Figure 23A] FIG. 23A shows experimental results from testing according to an embodiment of the present disclosure. [Figure 23B] FIG. 23B shows experimental results from testing according to an embodiment of the present disclosure. [Figure 24A] FIG. 24A shows experimental results from testing according to an embodiment of the present disclosure. [Figure 24B] FIG. 24B shows experimental results from testing according to an embodiment of the present disclosure. [Figure 25A] FIG. 25A shows experimental results from testing according to an embodiment of the present disclosure. [Figure 25B] FIG. 25B shows experimental results from testing according to an embodiment of the present disclosure. [Figure 26A] FIG. 26A shows experimental results from testing according to an embodiment of the present disclosure. [Figure 26B] FIG. 26B shows experimental results from testing according to an embodiment of the present disclosure. [Figure 27] FIG. 27 shows experimental results from testing according to an embodiment of the present disclosure.

[0016] Embodiments of the present disclosure will be described with reference to the accompanying drawings. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] While specific configurations and arrangements are described, it should be understood that this is done for illustrative purposes only. A person skilled in the art will recognize that other configurations and arrangements can be used without departing from the spirit and scope of the present disclosure. It will be apparent to one skilled in the art that the present disclosure can be employed in a variety of other applications.

[0018] Units, prefixes, and symbols are expressed in the form accepted by the International System of Units (SI). Numerical ranges are intended to be inclusive of the numbers defining the range. When a range of numerical values ​​is stated, it is understood that each integer and fraction between the numerical values ​​stated as the upper and lower limits of the range is also specifically disclosed, along with each subrange between the numerical values. Thus, ranges described herein are understood to be shorthand for all values ​​within the range, including the stated endpoints. For example, a range from 1 to 10 is understood to include every numerical value, combination of numerical values, or subranges in the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0019] Where a numerical value is explicitly recited, it is understood that numerical values ​​that are approximately the same amount or quantity as that numerical value are also within the scope of the disclosure. Where combinations are disclosed, subcombinations of each element of the combination are also specifically disclosed and are within the scope of the disclosure. Conversely, where different elements or groups of elements are individually disclosed, combinations thereof are also disclosed.

[0020] The use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination thereof. As used herein, the indefinite article "a" or "an" should be understood to refer to "one or more" of the described or listed components.

[0021] The term "about" refers to a value or composition that one of ordinary skill in the art would determine to be within an acceptable error range for a particular value or composition, depending on the method of measuring or determining the value or composition, i.e., the limitations of the measurement system. For example, "about" can mean within 1× standard deviation or more than 1× standard deviation, as is customary in the art. Alternatively, "about" can mean a range of up to 10%. Furthermore, particularly with respect to biological systems or processes, the term can mean up to an order of magnitude or up to 5-fold. When a particular value or composition is described in the present application and claims, unless otherwise indicated, the meaning of "about" should be assumed to be within an acceptable error range for that particular value or composition.

[0022] As used herein, concentration ranges, percentage ranges, ratio ranges, and integer ranges are understood to include any integer value within the stated range, and fractions thereof, where appropriate (such as tenths or hundredths of an integer), unless otherwise specified.

[0023] Introduction: The current standard of care in the detection and treatment of sepsis relies on blood cultures, with a median time to detection of approximately 13 hours. Blood culture tests detect microorganisms without pathogen identification (ID), and positive samples are subsequently plated in petri dishes. In the traditional blood culture process, two sets of blood cultures are taken per adult patient, with each set consisting of an aerobic and an anaerobic bottle to ensure that the full spectrum of sepsis-causing bacteria is captured during culture. Typically, each culture is taken from a separate venipuncture (e.g., the patient's left arm and right arm). This is to ensure that bacterial shedding events are captured by culture and that bacteria are "recovered" for subsequent testing (e.g., ID and AST). After incubation, the aerobic and anaerobic bottles are incubated in the blood culture machine and growth is monitored in real time. The aerobic and anaerobic bottles are incubated and agitated until the bacteria complete the lag-log growth transition, at which point they are electronically detected. Laboratory personnel may then be notified that the patient's culture is positive. Cultures typically become positive in an average of about 13 hours for most bacteria, but may take longer (up to 5 days) for yeasts and fungi. However, many cultures remain negative due to sampling errors, insufficient blood volume, delays in transport to the laboratory, or lack of sensitivity.

[0024] Due to the urgency of sepsis, after a positive test, the laboratory immediately performs a gram stain (e.g., gram-positive or gram-negative bacteria) to identify the key microorganism, contaminant, monomicrobial, or polymicrobial infection, and reports intermediate information to the healthcare provider. Additionally, the laboratory can take immediate measures to identify the bacteria using rapid methods such as molecular diagnostic systems, the results of which can be obtained in 1.5 hours. These systems may provide limited molecular information on the genetic drug resistance of specific bacteria that show a specific profile. Alternatively, the laboratory can process the positive blood culture (PBC) aliquot with a matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) mass spectrometry system and report the ID in about 1 hour. The healthcare provider can use this ID to confirm and possibly adjust antibiotics that may have been administered prophylactically to the patient. However, by the time the bacteria is identified, up to 20–24 hours (at best) may have passed since the patient's culture was first taken.

[0025] Concurrent with obtaining and reporting an identified pathogen, antibiotic susceptibility testing (AST) may be performed to determine the antibiotic susceptibility profile of the pathogen. In some cases, AST may be performed in vitro on isolated bacterial pathogens using manual methods such as broth microdilution or culture-based assays (e.g., antibiotic susceptibility testing by disk diffusion or using cultures inoculated in Petri dishes). In other cases, AST may be performed using automated equipment to determine the minimum inhibitory concentration (MIC) of resistance to an antimicrobial agent or drug.

[0026] However, these systems may require isolated bacterial colonies. For example, current AST methods may require large biomasses of clean bacteria to function properly. Subcultures from positive blood cultures must first be grown in inoculated media in petri dishes. This process can be completed in 6–12 hours in laboratories that operate 24 hours a day, but may take up to 24 hours in laboratories that are closed overnight. The culture may then need to be homogenized (e.g., inoculated with 10,000 cells) before inoculating the AST system. After inoculation, the AST analysis takes 8–16 hours to report drug susceptibility or resistance information for all bacterial taxa. In addition, AST may not begin until at least the second day after a patient is admitted to the hospital. This means that the results of the AST analysis (e.g., bacterial susceptibility, intermediate, or resistant (SIR), MIC, or resistance information for a particular antibiotic) may be reported to healthcare professionals 2.5–3 days after the initial blood culture sample is drawn from the patient.

[0027] The entire process of reporting critical antibiotic information for a septic patient can take several days. For example, when an infection is suspected, blood, urine, sputum, and other samples are taken from the patient and submitted to a clinical laboratory to first determine if an infectious agent is present. It may take 18-24 hours (e.g., day 1) for most pathogenic bacterial species to grow sufficiently. If bacteria are isolated, an additional 18-24 hours (e.g., day 2) is required to culture the isolate, and an additional 2-48 hours (e.g., day 3 or later) is required to identify the bacterial isolate and perform an AST. Traditional and automated AST methods require a "pure culture" of the bacterial isolate, followed by a lengthy incubation period (e.g., 6-24 hours) for bacterial growth. Traditional systems are growth-based and therefore limited in that they are time-consuming, expensive, manual, and non-integrated. Without remedial measures, patients may continue to suffer, and often worsen, as physicians treat with empirical antibiotics while waiting for more actionable information from the laboratory about the pathogen or pathogens (e.g., bacterial pathogens) causing the infection.

[0028] Current technology cannot provide a comprehensive solution that integrates the entire workflow of host response detection, pathogen identification, and antimicrobial or antibiotic susceptibility testing (AST). In some cases, some systems focus on only a single aspect of the sepsis cascade, such as host response detection or pathogen detection. For example, systems can examine host response as an early sign of sepsis by detecting molecular leukocyte RNA markers (via detection of gene expression by reverse transcription (RT)-PCR), but cannot provide answers regarding pathogen identification or susceptibility. Immune response results can alert healthcare professionals that a patient has entered or is entering the sepsis cascade and indicate the urgent need for treatment or intervention to prevent potential irreversible morbidity. Healthcare professionals identify the site of infection and infectious agent in a traditional manner, such as taking blood cultures from the site of infection to identify the infectious agent, and respond immediately.

[0029] In terms of pathogen detection, current technology offers detection and identification methods using PCR directly from blood samples. However, such systems are expensive, have limited menu options, are difficult to service, may not provide a solution for rapid AST results, and may only offer limited molecular genetic resistance panels. Other systems may utilize rRNA RT-PCR methods for pathogen identification that detect pathogens directly from blood, but may be constrained by a limited menu (e.g., 15 or fewer targets). Ultimately, current technology does not offer a solution for rapid automated AST directly from blood, instead relying on positive blood cultures that can take 13–20 hours for any useful result reporting. For example, some systems can obtain aliquots from positive blood culture bottles (PBCs), reducing the time (e.g., 6–24 hours) required to grow bacteria isolated from PBC bottles. However, these systems are limited in the types of drugs and bacteria they can report, limiting their usefulness to healthcare professionals. To significantly reduce morbidity and mortality, new diagnostic methods, devices, and systems are needed to rapidly determine antibiotic susceptibility and resistance of infectious sepsis-causing bacteria at the single-cell or low copy number level directly from blood samples, without the significant time delays associated with multiple culture steps (e.g., biological amplification) currently required by standard treatment methods.

[0030] Because septic conditions often go undetected in patients and can rapidly progress to life-threatening conditions, there is a clear need and demand for new comprehensive systems, devices, and methods to perform rapid testing of patient samples and provide guidance on appropriate antimicrobials to save lives and reduce antimicrobial resistance (AMR). The systems, devices, and methods described herein provide a holistic and systematic approach to determining antibiotic susceptibility and resistance of pathogens to recommend effective and appropriate treatments for patients.

[0031] AST System Overview: 1 illustrates a diagram of a system 101 for performing antimicrobial susceptibility testing (AST) according to an embodiment of the present disclosure. In some embodiments, the system 101 may be referred to herein as an AST system 101. The system 101 may include an analyzer 108, a sample container 111, a sample preparation cartridge 114, an AST cartridge 115, a processing device 116, and a number of databases 110 communicatively coupled via a network 112.

[0032] The analyzer 108 may be a point-of-care (POC) testing device that performs phenotypic AST of pathogens in a patient sample, which may be stored in a sample container 111. In some embodiments herein, the analyzer 108 may be referred to as an analytical device. In some embodiments, the sample in the sample container 111 may consist of whole blood, urine, sterile bodily fluid, or other sample obtained from a patient. In some embodiments, the analytical device 108 may receive the sample container 111 placed by a user or operator of the analytical device 108 into a corresponding drawer in a housing of the analytical device 108.

[0033] In addition to receiving sample containers 111, the analyzer 108 can also receive sample preparation cartridges 114 and AST cartridges 115, which can likewise be placed in corresponding drawers in the housing of the analyzer 108 by a user or operator of the analyzer 108. In some embodiments, various components and subsystems within the analyzer 108 can interface with the sample containers 111, sample preparation cartridges 114, AST cartridges 115, processing device 116, and / or database 110 to perform sample preparation and processing, enrichment and cleanup of pathogens in the sample, AST using microscopy and / or fluorescence methods.

[0034] In some embodiments, after receiving the sample container 111, a pipetting system in the analytical device 108 may transfer the sample from the sample container 111 to the sample preparation cartridge 114. In some embodiments, the sample preparation cartridge 114 may be a dedicated consumable with a receptacle configured to hold the sample and elements for performing sample processing. The sample preparation cartridge 114 may include a processing tube 113 having a septum disposed on or within the tube to protect its contents. The processing tube 113 may be configured to receive the sample from the sample container 111 through the septum by transferring the sample using a needle of the sample preparation cartridge 114. Once the sample is transferred to the processing tube 113, the sample may undergo sample concentration, lysis, enrichment, cleanup, and / or other processing steps.

[0035] In some embodiments, the sample container 111 is a blood collection tube. Blood collection tubes are available from a variety of vendors and may contain a variety of reagents for storing blood.

[0036] In some embodiments, the sample container 111 is a blood culture bottle. Blood culture bottles are available from a variety of vendors and contain a growth medium that encourages the growth of microorganisms. Blood culture bottles may also contain a resin that absorbs antibiotics to reduce their effect on the microorganisms in the sample. In some embodiments, the sample in the blood culture bottle is incubated for a period of time to allow pathogens to grow but not reach a growth plateau (positive).

[0037] After sample preparation and processing (e.g., including sample concentration, enrichment, and cleanup) using the elements and components of the sample preparation cartridge 114, the sample is transferred by a pipetting system in the analyzer 108 from the processing tube 113 in the sample preparation cartridge 114 to the AST cartridge 115. In some embodiments, the AST cartridge 115 may be a specialized consumable with multiple reaction wells configured to hold multiple aliquots of the enriched sample for performing AST. In some embodiments, the analyzer 108 may use microscopy (e.g., single cell microscopy, fluorescence microscopy, etc.) by exposing multiple aliquots of the enriched sample in the AST cartridge 115 to a predetermined concentration of an antimicrobial agent.

[0038] In some embodiments, the analytical device 108 may further interface with additional cartridges, such as a polymerase chain reaction (PCR) cartridge. In some embodiments, the PCR cartridge may be a specialized consumable with multiple reaction chambers configured to hold multiple aliquots of a sample for performing a nucleic acid amplification step to perform pathogen identification. In some embodiments, the analytical device 108 may be configured to simultaneously hold one or more sample preparation cartridges 114, PCR cartridges, and / or AST cartridges 115 for simultaneous or sequential sample preparation / processing, pathogen identification, and / or susceptibility testing. In some embodiments, the sample preparation cartridges 114, AST cartridges 115, and / or PCR cartridges may be referred to herein as consumables or containers configured for insertion into the analytical device 108.

[0039] In some embodiments, the analyzer 108 can include a controller 109 disposed within a housing of the analyzer 108. The controller 109 can control the movement and operation of different components within the analyzer 108, including the movement of one or more sample containers, processing tubes, cartridges, and pipetting systems within the analyzer 108. In some embodiments, the controller 109 can also control the operation of one or more centrifuges, subsystems, and modules within the analyzer 108 to perform sample preparation, pathogen identification, susceptibility testing, and / or other functions. In some embodiments, the controller 109 can include a microcontroller on an integrated circuit (IC) chip within the analyzer 108 that is programmed to turn on / off and operate one or more centrifuges, subsystems, and modules within the analyzer 108. In some embodiments, the controller 109 can be coupled to and programmed to control the operation of one or more stepper motors, actuators, or other motion control components within the analyzer 108.

[0040] In some embodiments, the controller 109 may be programmed by the processing unit 116. The processing unit 116 may be a computing device coupled to the analyzer 108 to perform data processing and provide instructions to the controller 109 and / or other components within the analyzer 108. In some embodiments, the processing unit 116 may be a personal digital assistant, a desktop workstation, a laptop or notebook computer, a netbook, a tablet, a smartphone, a mobile phone, a smartwatch, or any combination thereof.

[0041] In some embodiments, the processing unit 116 communicates with the analytical unit 108 to receive the results of the reactions that occurred in the AST cartridge 115 and can perform further processing and data analysis to determine the susceptibility of one or more pathogens in the sample. In some embodiments, the processing unit 116 receives one or more images of the AST cartridge 115 from the analytical unit 108 and compares the images to a control to determine whether a particular pathogen is susceptible, intermediate, or resistant to a particular antimicrobial agent.

[0042] In some embodiments, the processing unit 116 may also be in communication with the multiple databases 110. In some embodiments, one or more of the multiple databases 110 may represent any number of databases and may include various databases, such as those storing clinical parameter data, epidemiological information, or antibiotic resistance information for multiple pathogens. In some embodiments, one or more of the multiple databases 110 may be configured to store pathogen taxonomy data and / or results of past pathogen identification workflows (e.g., performed by the analyzer 108). In some embodiments, information from one or more databases 110 may be used to select antimicrobial agents to test using the AST cartridge 115 and the analyzer 108. In some embodiments, information from one or more databases 110 may be used to determine whether the tested pathogen is resistant, intermediate, or susceptible to an agent from the results of testing by the AST system 101. In some embodiments, one or more of the multiple databases 110 may be configured to store predefined rules for making antimicrobial susceptibility determinations for various pathogens. In some embodiments, the processing device 116 may use at least one of the pathogen classification data in the database 110, the results, and / or the predefined rules to report susceptibility information to a user of the analysis device 108. In some embodiments, the processing device 116 may not report a particular drug susceptibility for a pathogen based on identifying from an analysis of the predefined rules that the pathogen is not susceptible to a particular drug. For example, Klebsiella is naturally resistant to ampicillin, so the processing device 116 may not report this susceptibility information to the user.

[0043] In some embodiments, one or more of the plurality of databases 110 may include electronic health record (EHR) data including patient health care information obtained from various health care services and health care providers, such as hospitals, clinical care facilities, laboratories, radiology departments, pharmacies, etc. In some embodiments, the EHR data stored in the databases 110 includes patient and medical history data regarding the health and treatment of the patients, including demographics, medical history, medications and allergies, immunization status, laboratory test results, radiology images, vital signs, personal statistics such as age and weight, and billing information for each patient. In some embodiments, the processing device 116 may use the results of the pathogen identification and / or AST performed by the analysis device 108 along with the data stored in the plurality of databases 110 (e.g., clinical parameter data, epidemiological or antibiotic resistance information, EHR data, etc.) to determine treatment recommendations for the patient.

[0044] In some embodiments, the components in the system 101 may be communicatively coupled via a network 112. In particular, the network 112 may enable the transmission and communication of information between the analytical device 108, the multiple databases 110, the processing device 116, and / or any other devices or components in the system 101. In some embodiments, the system 101 may include additional components, such as a Raman spectroscopy device and / or an electronic health record (EHR) system (not shown).

[0045] In some embodiments, the network 112 may be any or a combination of a LAN (local area network), a WAN (wide area network), a telephone network, a wireless network, a point-to-point network, a star network, a token ring network, a hub network, or any other suitable configuration. The network may conform to one or more network protocols (including IEEE (Institute of Electrical and Electronics Engineers) protocols, 3GPP (3rd Generation Partnership Project) protocols, Fourth Generation Wireless Protocols (4G) (e.g., LTE (Long Term Evolution) standards, LTE Advanced, LTE Advanced Pro), Fifth Generation Wireless Protocols (5G), and / or similar wired and / or wireless protocols) and may include one or more intermediate devices for routing data between the analysis device 108, the multiple databases 110, the processing device 116, and / or any other devices or components in the system 101.

[0046] Embodiments of the analysis device: FIG. 2 shows a diagram of an analytical device 200 according to an embodiment of the present disclosure. The analytical device 200 represents an exemplary embodiment of the analytical device 108 shown in FIG. 1. In some embodiments, the analytical device 200 may be referred to herein as an analyzer 200. The analytical device 200 is a benchtop device with a housing 201 that houses various components and modules for sample preparation, processing, and testing. In some embodiments, the housing 201 may include the body of the analytical device 200 and / or an exterior case of the analytical device 200 that protects the internal modules and components. In some embodiments, the analytical device 200 may be configured in a cubic, cuboid, or rectangular shape with various compartments for user access and manipulation of the analytical device 20 ... 3 It may have a compact size with dimensions less than 10 mm.

[0047] In some embodiments, the analytical device 200 may be coupled to a computing device (e.g., processing device 116), such as a personal digital assistant, desktop workstation, laptop or notebook computer, netbook, tablet, smartphone, mobile phone, smartwatch, or any combination thereof. A user or operator of the analytical device 200 can use the computing device to control the analytical device 200, to send and receive sample information, patient information, pathogen information, antimicrobial information, etc. to the analytical device 200, and to access and edit pathogen identification results from the analytical device 200.

[0048] 3A shows a front view of an analytical device 200 according to an embodiment of the present disclosure. FIG 3A shows the internal features located within the housing 201 of the analytical device 200, including a first pipettor 202, a second pipettor 204, a sample drawer 210, a sample cartridge drawer 220, and a processing cartridge drawer 230.

[0049] In some embodiments, the first pipettor 202 and the second pipettor 204 may be pipettor devices configured to handle liquid transfer between components within the housing 201 of the analytical device 200. In some embodiments, the first and second pipettors 202, 204 may be automated devices controlled by a controller 109 in the analytical device 200. In some embodiments, the controller 109 may control the movement of the first and second pipettors 202, 204, including vertical and / or horizontal movement of the first and second pipettors 202, 204 relative to different components within the analytical device 200. In some embodiments, there may be more or fewer pipettors in the analytical device 200.

[0050] In some embodiments, the first pipettor 202 and the second pipettor 204 may be referred to as a high-volume pipettor and a low-volume pipettor, respectively. In some embodiments, the first pipettor 202 may be configured to handle volumes ranging from about 50 microliters (μL) to 5 milliliters (mL), while the second pipettor 204 may be configured to handle volumes ranging from about 1 to 200 μL. In some embodiments, the first pipettor 202 may have a coefficient of variation (CV) of 5% at 50 μL and a CV of 1% at 5 mL, and the second pipettor 204 may have a CV of 5% at 1 to 200 μL.

[0051] In some embodiments, the first and second pipettors 202, 204 may be referred to herein as pipettors and / or pipettor systems. In some embodiments, the first and second pipettors 202, 204 may each be configured to be attached to a removable, disposable needle. The first and second pipettors 202, 204 may be attached to two different needles configured to handle different volume ranges as required by the first and second pipettors 202, 204.

[0052] In addition to transferring liquids, the first and second pipettors 202, 204 may be configured to move elements, such as tubes, cartridges, etc., within the housing 201 of the analytical device 200. In particular, the first and second pipettors 202, 204 may each be configured with a pipette tip holder that can be attached to various components used in the analytical device 200. In particular, the pipette tips of the first and second pipettors 202, 204 can be pressed into and engaged with handling features, such as needles, tubes, cartridges, spin column baskets, etc., to pick up and place various components in different modules or areas within the analytical device 200.

[0053] In some embodiments, the first and second pipettors 202, 204 may have dimensions of approximately 325 mm (width) x 575 mm (depth) x 435 mm (height). In some embodiments, the first and second pipettors 202, 204 may be located in an upper section of the housing 201 such that the first and second pipettors 202, 204 interact with samples, tubes, cartridges, and different modules in a lower section of the housing 201. In particular, the first and second pipettors 202, 204 may handle liquid transfer and component movement in the sample drawer 210, sample cartridge drawer 220, and processing cartridge drawer 230 shown in FIG. 3A.

[0054] In some embodiments, the sample drawer 210, the sample cartridge drawer 220, and the processing cartridge drawer 230 are configured as sliding horizontal compartments designed to fit within three corresponding receptacles in the housing 201 of the analytical device 200. In some embodiments, the sample drawer 210, the sample cartridge drawer 220, and the processing cartridge drawer 230 may be configured to receive specialized elements that are inserted into the analytical device 200 for sample processing and testing. In particular, the sample drawer 210 can receive a sample container (e.g., sample container 111) that contains a sample obtained from a patient. The sample cartridge drawer 220 can receive a sample preparation cartridge (e.g., sample preparation cartridge 114) to which the sample is transferred by components within the analytical device 200.

[0055] In some embodiments, the processing cartridge drawer 230 can receive an AST cartridge (e.g., AST cartridge 115) configured to receive an aliquot of enriched sample after processing and enrichment of the sample in the sample preparation cartridge. In additional or alternative embodiments, the processing cartridge drawer 230 may be used as an AST cartridge and / or a PCR cartridge drawer. For example, a PCR cartridge or an AST cartridge may be inserted into the processing cartridge drawer 230 depending on whether the analytical device 200 is being used to perform pathogen identification or antimicrobial susceptibility testing of the sample. In some embodiments, the analytical device 200 may be configured to perform both pathogen identification and antimicrobial susceptibility testing functions with the dual processing cartridge drawer 230 configured to interface with dedicated cartridges or consumables for AST and pathogen identification.

[0056] In some embodiments, the sample drawer 210, the sample cartridge drawer 220, and the processing cartridge drawer 230 may include readers configured to scan identifiers of the sample containers, sample preparation cartridges, and AST cartridges (or PCR cartridges), respectively. In some embodiments, a reader in the drawer may scan the identifiers of the sample containers and / or cartridges as each drawer is inserted into the housing 201 of the analytical device 200. In some embodiments, the readers in the drawers 210, 220, and 230 may be configured to scan identification codes, bar codes, or data matrices of the corresponding sample containers and / or cartridges. In some embodiments, the readers in the drawers 210, 220, and 230 may be bar code readers, quick response (QR) code readers, or the like.

[0057] In some embodiments, FIG. 3A illustrates additional components disposed within housing 201 including one or more centrifuges configured to process tubes and cartridges for sample processing.

[0058] 3B is a diagram illustrating a top view of an analytical device 200 according to an embodiment of the present disclosure. FIG. 3B shows a cross-sectional view from the top view of the analytical device 200, illustrating various components, modules, and / or subsystems located within the housing 201 of the analytical device 200 beneath the first and second pipettors 202, 204.

[0059] 3B includes a sample drawer 210 containing a plurality of sample vessels 212, a sample cartridge drawer 220 containing a plurality of sample preparation cartridges 224, and a processing cartridge drawer 230 containing both an AST cartridge 232 and a PCR cartridge 234. The sample vessels 212, sample preparation cartridges 224, and AST cartridges 232 represent exemplary embodiments of the sample vessels 111, sample preparation cartridges 114, and AST cartridges 115, respectively, shown in FIG.

[0060] In some embodiments, the processing cartridge drawer 230 may be configured to house both AST cartridges 232 and PCR cartridges 234 for performing pathogen identification and antimicrobial susceptibility testing. In some embodiments, each drawer in the housing 201 may house a predetermined number of sample containers 212, sample preparation cartridges 224, AST cartridges 232, and / or PCR cartridges 234 at one time. In some embodiments, the sample preparation cartridges 224, AST cartridges 232, and / or PCR cartridges 234 may be disposable after a single use or may be reused for testing additional samples.

[0061] FIG. 3B also shows a first centrifuge 240 , a second centrifuge 242 , a PCR subsystem 244 , an enrichment subsystem 246 , a mechanical device 248 , and an AST subsystem 250 disposed within the housing 201 .

[0062] In some embodiments, the first centrifuge 240 may be a high-speed centrifuge configured to centrifuge the processing tubes (e.g., processing tubes 113) and / or spin column baskets placed in the first centrifuge 240 by the first pipettor 202. In some embodiments, the second centrifuge 242 may be a low-speed centrifuge configured to centrifuge the AST cartridges 232 placed in the second centrifuge 242 by the second pipettor 204. In some embodiments, the first and second centrifuges 240, 242 may centrifuge the processing tubes, spin column baskets, and / or AST cartridges 232 in a swinging bucket configuration. In some embodiments, the first and second centrifuges 240, 242 may configure a cylindrical shape with a diameter of about 250 mm and a height of about 175 mm.

[0063] In some embodiments, the PCR subsystem 244 may include a thermal cycler configured to control temperature in performing quantitative PCR (qPCR) and an optical system for optical interrogation of reaction chambers in the PCR cartridge by fluorescence. In some embodiments, the thermal cycler of the PCR subsystem 244 may control temperatures in the range of about 35° C. to 100° C. In some embodiments, the optical system of the PCR subsystem 244 may perform fluorescence measurements from the bottom of the PCR cartridge in the housing 201. In some embodiments, the PCR cartridge may be placed in and removed from the PCR subsystem 244 by the second pipettor 204. In some embodiments, the PCR subsystem 244 may hold up to two PCR cartridges at the same time, where the PCR cartridges undergo thermal cycling independently within the PCR subsystem 244. In some embodiments, the dimensions of the PCR subsystem 244 are approximately 70 mm (width) by 125 mm (depth) by 250 mm (height).

[0064] In some embodiments, the enrichment subsystem 246 may be configured to hold multiple processing tubes and apply a swinging motion to the processing tubes to allow vibration of the sample and mixing of pathogens in the sample with the growth medium for pathogen growth and enrichment. In some embodiments, the first pipettor 202 may be configured to vertically load the tubes into the slots of the enrichment subsystem 246. In some embodiments, the enrichment subsystem 246 reorients the tubes from a vertical position to a horizontal position and applies a swinging motion of ±15° about the horizontal position at a frequency of 1 Hz. In some embodiments, the enrichment subsystem 246 can hold up to about four 15 mL processing tubes at a time, with each processing tube representing a different sample.

[0065] In some embodiments, the enrichment subsystem 246 may include a moving magnet. The moving magnet may engage the processing tube when in a vertical position within the enrichment subsystem 246. In some embodiments, the enrichment subsystem 246 may apply a 37° C. temperature control of the processing tubes held within the enrichment subsystem 246. In some embodiments, the dimensions of the enrichment subsystem 246 are approximately 150 mm (width) by 175 mm (depth) by 115 mm (height).

[0066] In some embodiments, the mechanical device 248 may be configured to agitate the processing tube to effect lysis of microorganisms in the sample. In some embodiments, the first pipettor 202 may be configured to vertically load the tube into a slot of the mechanical device 248. In some embodiments, the mechanical device 248 may include an agitator device or cell disrupting device configured to apply a high-speed vibrational motion to the processing tube. In some embodiments, the mechanical device 248 may apply the high-speed vibrational motion by applying a reciprocating motion along a predetermined axis by the agitator device while the processing tube is in a vertical position.

[0067] In some embodiments, the mechanical device 248 can hold up to two process tubes at a time and apply ±2° angular motion to the process tubes. In some embodiments, the mechanical device 248 can vibrate the process tubes at about 5,000-30,000 cycles per minute. In some embodiments, the dimensions of the mechanical device 248 can be about 75 mm (width) x 175 mm (depth) x 100 mm (height).

[0068] In additional or alternative embodiments, the mechanical device 248 may include a sonicator configured to sonicate the process tube to agitate the sample.

[0069] In some embodiments, the AST subsystem 250 may include a heater configured for incubation of samples in the AST cartridge 232 in the housing 201 and an imaging subsystem for imaging the reaction wells of the AST cartridge 232. In some embodiments, the heater of the AST subsystem 250 may be used for temperature control of the incubation of samples in the AST cartridge 232. In some embodiments, the imaging subsystem of the AST subsystem 250 may include a microscope configured to scan the bottom of each reaction well of the AST cartridge 232 using a motorized XYZ translation stage to acquire images. In some embodiments, the imaging subsystem of the AST subsystem 250 may include a fluorescent sensor with two optical channels for detecting different fluorescent signals (e.g., green and red).

[0070] In some embodiments, the AST subsystem 250 can identify antimicrobial phenotypic resistance of a microorganism (e.g., a pathogen) based on the acquired images and / or signals. In some embodiments, the AST cartridge 232 can be placed into and removed from the AST subsystem 250 by the second pipettor 204.

[0071] In some embodiments, the AST subsystem 250 can hold up to five AST cartridges 232 at a time. In some embodiments, the AST subsystem 250 can apply a C temperature control, such as by using a thermal block, to the AST cartridges 232 held within the AST subsystem 250. In some embodiments, the AST subsystem 250 may apply a temperature control of approximately 37° C. In some embodiments, the dimensions of the AST subsystem 250 may be approximately 200 mm (width) by 185 mm (depth) by 285 mm (height).

[0072] FIG. 4 shows a diagram of an analytical device 200 with three open compartments, according to an embodiment of the present disclosure. In some embodiments, the analytical device 200 of FIG. 4 shows the sample drawer 210, the sample cartridge drawer 220, and the processing cartridge drawer 230 extending from the housing 201 in an open position for loading and / or removing samples and / or cartridges. The sample drawer 210, the sample cartridge drawer 220, and the processing cartridge drawer 230 may be pushed into the housing 201 in a closed position, where the compartments fit into three corresponding receptacles in the housing 201. In some embodiments, the opening and closing of the sample drawer 210, the sample cartridge drawer 220, and the processing cartridge drawer 230 may be controlled by a controller (e.g., controller 109) and / or the processing device 116 of the analytical device 200. In some embodiments, a user of the processing device 116 can control the opening and closing of the sample drawer 210, the sample cartridge drawer 220, and the processing cartridge drawer 230 using software installed on the processing device 116. In some embodiments, the sample drawer 210, the sample cartridge drawer 220, and the processing cartridge drawer 230 can be manually pulled out and pushed into the housing 202 by a user to load and / or unload sample containers, cartridges, and / or other elements into the analysis device 200.

[0073] In some embodiments, the sample drawer 210 can hold multiple samples. In some embodiments, the sample drawer 210 can hold up to 10 samples at a time, including samples stored in sample containers 212. In some embodiments, the sample containers 212 shown in FIG. 4 can represent one or more blood sample tubes, urine sample tubes, and / or blood culture bottles. In some embodiments, the sample cartridge drawer 220 can accommodate multiple sample preparation cartridges, for example, up to 10 sample preparation cartridges at a time. In some embodiments, the processing cartridge drawer 230 can accommodate multiple processing cartridges, for example, up to 12 PCR cartridges, 6 AST cartridges, or a combination thereof at a time.

[0074] In some embodiments, the dimensions of the sample drawer 210 may be approximately 40 mm (width) x 155 mm (depth) x 600 mm (height). In some embodiments, the dimensions of the sample cartridge drawer 220 may be approximately 85 mm (width) x 150 mm (depth) x 615 mm (height). In some embodiments, the dimensions of the processing cartridge drawer 230 may be approximately 140 mm (width) x 155 mm (height) x 270 mm (depth).

[0075] Examples of sample processing, concentration, enrichment, and cleanup in analytical instruments include: In some specific examples, components located within the analyzer 200 may perform sample processing, concentration, enrichment, and clean-up steps in the processing tube 113 before performing the AST assay and acquiring an image of the sample in the AST cartridge 232. In some embodiments, the first pipettor 202 of the analyzer 200 transfers the sample from the sample container 111 to the processing tube 113 and performs steps to process, separate, concentrate, and enrich for rapid detection of pathogens in the sample.

[0076] In some embodiments, the sample processing may include a step of blood cell lysis. In embodiments where the sample is a blood sample, the first pipettor 202 may be configured to perform blood cell lysis of the blood sample. The first pipettor 202 may add one or more lysis reagents to the processing tube 113. The one or more lysis reagents may be mixed with the blood sample in the processing tube 117 using a mixer (e.g., enrichment subsystem 246, etc.) disposed in the housing 201 of the analyzer 200 to lyse blood cells in the blood sample. In some embodiments, the one or more lysis reagents may include one or more saponin-based buffers. In some embodiments, the one or more lysis reagents may include one or more detergents, surfactants, or proteases.

[0077] After lysis of blood cells in the blood sample, the first pipettor 202 may transfer the processing tube 117 to a centrifuge 240 (or 1402 in FIG. 14A) for concentration and enrichment of the lysed sample. Concentration and enrichment of the sample may involve a series of steps using components in the analyzer 200, including centrifugation. In some embodiments, the first pipettor 202 in the analyzer 200 may transfer the processing tube 117 to a centrifuge, such as the first centrifuge 240 or 1402. In some embodiments, the centrifuge 240 or 1402 applies centrifugal force to the processing tube 117 to concentrate pathogens in the sample, for example, by separating the pathogens from other components in the sample.

[0078] After centrifugation, the first pipettor 202 may be used to remove liquid from the processing tube 117, leaving the concentrated pathogens in the processing tube 117. In some embodiments, the first pipettor 202 may then be used to add growth media to the concentrated pathogens in the processing tube 117, allowing the concentrated pathogens in the processing tube 117 to grow for a predetermined period of time. In some embodiments, the predetermined period of time is a period that allows for growth of the concentrated pathogens, for example, about 4 hours or more. In some embodiments, the predetermined period of time is 3-4 hours for enrichment of most pathogens, although other pathogens may require a longer period of time for growth / enrichment. In some embodiments, the growth media is stored in one or more reservoirs of the sample preparation cartridge 224. In some embodiments, the growth media may include Mueller Hinton Broth, cation-adjusted Mueller Hinton Broth, tryptic soy broth, lysogeny broth, brain heart infusion (BHI) broth, and the like.

[0079] After concentration and enrichment, the analyzer 200 may perform a sample washing or clean-up step to obtain the pathogens in the enriched sample. In some embodiments, to wash the enriched pathogens, the first pipettor 202 moves the processing tube 117 to a centrifuge 240 or 1402 after a predetermined period of growth (after adding growth medium) and removes the supernatant from the processing tube 117 to obtain the enriched sample.

[0080] In some embodiments, the analyzer 200 may perform a sample clean-up step by immunomagnetic separation (IMS) techniques, such as using magnetic beads. In some embodiments, the processing tube 117 may include a plurality of magnetic beads configured to attach to the concentrated pathogens in the processing tube 117. In some embodiments, the magnetic beads may be coated with non-specific ligands. In some embodiments, the magnetic beads are coated with specific ligands dedicated to a particular pathogen or group of pathogens of the concentrated pathogens in the processing tube 117. In some embodiments, the pathogens are immobilized on the surface of the magnetic beads after an incubation period and can be concentrated into a pellet using a magnetic field.

[0081] In some embodiments, the first pipettor 202 can move the processing tube 117 to a magnet station, such as the enrichment subsystem 246, and apply a magnetic force to the processing tube 117 to retain the enriched pathogens attached to the magnetic beads. In some embodiments, the first pipettor 202 can remove excess liquid from the processing tube 117 after retaining the enriched pathogens attached to the magnetic beads in the processing tube 117. Removal of the excess liquid can result in an enriched sample containing the enriched pathogens.

[0082] In some embodiments, the concentrated pathogens may be resuspended and washed to remove debris or other materials in a wash step. In particular, the first pipettor 202 may add one or more wash materials to the concentrated pathogens in the processing tube 117 to wash and remove blood components or debris from the concentrated pathogens, leaving the enriched sample in the processing tube 117. In some embodiments, the one or more wash materials may include a combination of one or more buffers, detergents, surfactants, and proteases. In some embodiments, the one or more wash materials may be stored in the sample preparation cartridge 224.

[0083] In some embodiments, the analyzer 200 may be configured to identify the number of pathogens in the sample. In particular, the analyzer 200 may use one or more fluorescent dyes and a microscope (e.g., AST subsystem 250) to label cells in the sample and count the number of pathogens. In some embodiments, the number of pathogens in the initial sample (e.g., before concentration and enrichment) may be less than 200. By performing the concentration and enrichment steps, the number of pathogens in the enriched sample may increase to a range of about 1,000 to about 100,000. In some embodiments, the number of pathogens in the enriched sample may be about 10,000. In response to identifying the number of pathogens in the sample, the analyzer 200 may be configured to further enrich or dilute the sample to obtain a predetermined number of pathogens in the enriched sample.

[0084] In some embodiments, the sample may be treated with a protease and / or DNAse before or after enrichment. For example, the protease and / or DNAse may be added to the sample after enrichment but before enrichment. In another example, the protease and / or DNAse may be added to the sample at the same time as the lysis reagent.

[0085] In some embodiments, the sample may be treated with protease and / or DNAse during the clean-up process. For example, protease and / or DNAse may be added during the immunomagnetic capture process to digest and reduce the amount of debris in the sample. In another example, protease and / or DNAse may be added after the immunomagnetic capture process, which has the advantage of avoiding the protease / DNAse reagent from interfering with the immunomagnetic separation process. This is particularly important when the capture of bacteria by the beads is based on proteins that can be degraded by proteases, which may prevent its effectiveness.

[0086] After concentration, enrichment, and cleanup, the enriched sample in the processing tube 117 may then be ready to be transferred by a pipetting system in the analyzer 200 to the AST cartridge 232 for AST assay and image acquisition to determine the susceptibility of the enriched pathogens in the enriched sample.

[0087] Sample Preparation Cartridge Embodiments: FIG. 5 shows a diagram of a sample preparation cartridge 500 according to an embodiment of the present disclosure. The sample preparation cartridge 500 represents an exemplary embodiment of the sample preparation cartridge 224 shown in FIG. 3B. The sample preparation cartridge 500 may be a consumable item that is inserted into the sample drawer 210 of the analytical device 200 to prepare and process samples in sample containers. In some embodiments, the sample preparation cartridge 500 may be manufactured by injection molding from a polypropylene (PP) material. The sample preparation cartridge 500 may include a housing 502, a plurality of reservoirs 504, a lid 506, and an identifier 508.

[0088] In some embodiments, the housing 502 may have an elongated rectangular shape with rounded edges. The housing 502 may be configured to hold additional elements used in sample preparation, as shown in FIG. 6A. In some embodiments, the multiple reservoirs 504 may be separate reservoirs or tubes molded together. The multiple reservoirs 504 may be configured to store materials used to perform sample concentration, lysis, and / or nucleic acid amplification. In some embodiments, the materials stored in the reservoirs 504 may include one or more buffers (e.g., a salt-based buffer, a phosphate-buffered saline (such as PBS), a surfactant, a protease, a growth medium, etc.). In some embodiments, the reservoirs 504 may store one or more lysis reagents, such as one or more saponin-based buffers, surfactants, or proteases for performing cell lysis of the blood sample. In some embodiments, the reservoirs 504 may store one or more wash materials, which may include a combination of one or more buffers, surfactants, and proteases.

[0089] In some embodiments, the lid 506 of the sample preparation cartridge 500 is a protective lid that extends over the housing 502 and the multiple reservoirs 504. In some embodiments, the identifier 508 is an identifier of the sample preparation cartridge 500 that can be scanned by the analysis device 200 to perform sample preparation for antimicrobial susceptibility testing. In some embodiments, the identifier 508 may be at least one of an identification code, a bar code, or a data matrix such as a QR code. In some embodiments, the dimensions of the sample preparation cartridge 500 may be approximately 80 mm (width) x 55 mm (depth) x 155 mm (height), as shown in FIG. 5.

[0090] 6A shows an exploded perspective view of a sample preparation cartridge 500 with a processing tube 510 and other components inserted therein, according to an embodiment of the present disclosure. The sample preparation cartridge 500 may include a processing tube 510, a first removable needle 512, and two second removable needles 514 housed in corresponding receptacles within a housing 502 of the sample preparation cartridge 500. The processing tube 510 represents an exemplary embodiment of the processing tube 113 shown in FIG.

[0091] In some embodiments, the processing tube 510 may comprise, for example, a 15 ml tube with a conical bottom. In some embodiments, the processing tube 510 may be removable from the sample preparation cartridge 500 for processing in other modules within the analytical device 200. In some embodiments, the processing tube 510 may be a tube to which a sample is transferred after the sample container and sample preparation cartridge 500 are loaded into the sample drawer 210 and sample cartridge drawer 220 of the analytical device 200, respectively.

[0092] In some embodiments, a sample may be transferred from a sample container in the sample drawer 210 to a processing tube 510 of the sample preparation cartridge 500 in the sample cartridge drawer 220 by a first removable needle 512. In some embodiments, the first pipettor 202 is adapted to be attached to the first removable needle 512 and configured to transfer the sample to and / or from the processing tube 510 by inserting the first removable needle 512 through a septum of the processing tube 510. In some embodiments, two second removable needles 514 are used for processing small volumes and the second pipettor 204 may be configured to be attached to the two second removable needles 514.

[0093] 6A also shows the openings 520 of the plurality of reservoirs 504 of the sample preparation cartridge 500. In some embodiments, the sample preparation cartridge 500 has 12 reservoirs 504, with each reservoir 504 holding a volume of approximately 2.5 mL. In some embodiments, the sample preparation cartridge 500 may include a pierceable film 516 covering a receptacle of the housing 502 and / or a foil seal 518 covering the openings 520 of the plurality of reservoirs 504. In some embodiments, the pierceable film 516 may be a polyester film and the foil seal 518 may include aluminum foil, and both the pierceable film 516 and the foil seal 518 are pierceable by the first and / or second removable needles 512, 514.

[0094] 6B shows an exploded perspective view of a sample preparation cartridge 500' with a processing tube 510 and other components inserted therein, according to an embodiment of the present disclosure. Sample preparation cartridge 500' is similar to sample preparation cartridge 500, but with a more linear shape and a peelable film 506' in place of lid 506.

[0095] Processing tube and needle embodiments: 7A and 7B show views of a process tube 700 according to an embodiment of the present disclosure. Process tube 700 represents an exemplary embodiment of process tube 510 shown in FIG. 6A. In particular, FIG. 7A shows process tube 700 after assembly, while FIG. 7B shows an exploded view of the components within process tube 700. Process tube 700 includes cap 702, septum 704, and tube 706.

[0096] In some embodiments, septum 704 may be secured inside tube 706 with cap 702 attached over septum 704 of process tube 700. In some embodiments, septum 704 may be configured to allow insertion and removal of needles (e.g., needles 512 and 514) for transfer of liquids into process tube 700 without having to remove cap 702 from tube 706. In some embodiments, septum 704 provides an airtight seal within process tube 700 to prevent contamination of the contents of process tube 700.

[0097] In some embodiments, the process tube 700 may include a handling structure at the top of the process tube 700 that is compatible for handling by a pipettor (e.g., the first and / or second pipettors 202, 204). In some embodiments, the handling structure of the process tube 700 may be a cylindrical cavity in the cap 702 that is compatible for insertion by the tip of a pipettor. In some embodiments, the pipette tips of the first and second pipettors 202, 204 can be pressed into and fitted into the cylindrical cavity of the cap 702 to allow the process tube to be picked up and moved within the analyzer 200.

[0098] In some embodiments, the cap 702 may be made from a high density polyethylene (HDPE) material and the tube 706 may be made from a polypropylene (PP) material. In some embodiments, the septum 704 may include at least one of rubber, polytetrafluoroethylene (PTFE), thermoplastic elastomer (TPE), silicone, butyl rubber, or a combination thereof. In some embodiments, the septum 704 may include a double layer of polytetrafluoroethylene (PTFE) and another material selected from the group consisting of silicone, rubber, and butyl rubber. In some embodiments, the dimensions of the assembled process tube 700 may comprise, for example, a height of about 110 mm and a diameter of about 20 mm. In some embodiments, the septum 704 may have a thickness in the range of about 1 mm to 2 mm.

[0099] In some embodiments, the processing tube 700 may include a plurality of magnetic beads configured to attach to the concentrated pathogens in the processing tube 700. In some embodiments, the plurality of magnetic beads may be coated with a non-specific ligand. In some embodiments, the plurality of magnetic beads may be coated with a specific ligand specific for the concentrated pathogens in the processing tube 700. In additional or alternative embodiments, the plurality of magnetic beads may be stored in one or more reservoirs 504 of the sample preparation cartridge 500.

[0100] In some embodiments, the process tube 700 may be configured to receive a needle, as shown in Figures 8A, 8B, and 8C, which show diagrams of a needle 800 configured for insertion into the process tube 700 according to some embodiments of the present disclosure.

[0101] In particular, FIG. 8A shows a needle 800 comprising a plastic body 810, a cannula 820, and a number of slots 825. In some embodiments, the plastic body 810 may be attached to the cannula 820 by bonding. In some embodiments, a pipettor (e.g., the first pipettor 202) in the analytical device may be attached to a proximal end of the plastic body 810 of the needle 800. In some embodiments, the plastic body 810 of the needle 800 includes an aerosol filter configured to prevent contamination of the pipettor in the analytical device. In some embodiments, the needle 800 may be a ventilating needle configured to ventilate the process tube 700 upon insertion of the needle. In some embodiments, ventilating the needle 800 facilitates relieving pressure within the sealed process tube 700. In some embodiments, the plastic body 810 of the needle 800 includes a number of slots 825 configured to provide an air communication path between the interior and exterior of the process tube 700 when the needle 800 is inserted through the septum 704 and into the tube 706. For example, four slots 825 may be provided in the injection portion of needle 800 that holds cannula 820. In some embodiments, slots 825 may be produced by injection molding.

[0102] 8B shows needle 800 during insertion into process tube 700, and FIG. 8C shows needle 800 after full insertion into process tube 700. In some embodiments, cannula 820 of needle 800 may be inserted through septum 704, into cap 702, and into tube 706 of process tube 700. In some embodiments, the distal end of plastic body 810 may fit into cap 702 of process tube 700 when cannula 820 is fully inserted into tube 706.

[0103] 9A and 9B show diagrams of a high-volume needle 900 and a low-volume needle 910, respectively, according to embodiments of the present disclosure. In some embodiments, the high-volume needle 900 and the low-volume needle 910 may be coupled to a first pipettor 202 and a second pipettor 204, respectively, within an analytical device 200.

[0104] 9A, the high-volume needle 900 may include a plastic body 902 and a cannula 904. In some embodiments, the plastic body 902 may be a reservoir configured to hold a volume of about 50 μL to 5 mL during transfer of liquid in an analytical device. In some embodiments, the plastic body 902 may include a filter 903 disposed therein to prevent contamination of a pipettor coupled to the high-volume needle 900.

[0105] In some embodiments, the plastic body 902 may be made of a polypropylene (PP) material. In some embodiments, the plastic body 902 of the needle 900 may have a diameter of about 16 mm and a length of about 50 mm. In some embodiments, the cannula 904 may be made of stainless steel. In some embodiments, the cannula 904 may have a length of about 100 mm. In some embodiments, the overall length of the plastic body 902 and the cannula 904 when assembled may be about 150 mm. In some embodiments, the high volume needle 900 may be a 17 gauge needle with an inner diameter (ID) of about 1.05 mm, an outer diameter (OD) of about 1.60 mm, and a cannula diameter (CD) of about 2.50 mm.

[0106] In some embodiments, the cannula 904 may further include a secondary cannula disposed about the inner core of the needle 900. In some embodiments, the cannula 904 includes one or more vent holes 906. In some embodiments, the cannula 904 of the needle 900 may be in fluid communication with the vent holes 906. In some embodiments, the cannula 904 may be a slotted cannula that includes a slot around the needle.

[0107] As shown in FIG. 9B, the low-volume needle 910 may include a plastic body 912 and a needle shaft 914. In some embodiments, the plastic body 912 may be a reservoir configured to hold a volume of about 1-200 μL during transfer of liquid in an analytical device. In some embodiments, the plastic body 912 may include a filter 913 disposed therein to prevent contamination of a pipettor coupled to the low-volume needle 910. In some embodiments, the plastic body 912 may be manufactured from a polypropylene (PP) material. In some embodiments, the plastic body 912 of the needle 910 may have a diameter of about 7.25 mm and a length of about 45 mm. In some embodiments, the needle shaft 914 may have a length of about 10 mm. In some embodiments, the needle shaft 914 may be made of stainless steel. In some embodiments, the low-volume needle 910 may be a 29 gauge needle with an inner diameter (ID) of about 0.20 mm and an outer diameter (OD) of about 0.30 mm.

[0108] FIG. 10 shows a diagram of an example high volume needle 900 according to an embodiment of the present disclosure. In particular, FIG. 10 shows various examples of the cannula of the high volume needle 900 with vent holes, slots, etc. In some embodiments, the first needle shown in FIG. 10 can be vented by having two communicating orifices or vent holes in the needle cannula. In some embodiments, the second and fourth needles shown in FIG. 10 can be vented when the tip of each needle is inserted into a septum (e.g., septum 704) of a processing tube.

[0109] FIG. 11 illustrates how a low-volume needle 910 interfaces with a sample preparation cartridge 500, according to embodiments of the present disclosure. In particular, FIG. 11 illustrates an example of a low-volume needle 910 disposed in one of the reservoirs 504 of the sample preparation cartridge 500, for use, for example, to transfer materials used for sample enrichment and / or lysis from the reservoir 504 to a sample in a processing tube. In some embodiments, the sample preparation cartridge 500 may be configured to receive one needle, such as the low-volume needle 910. In other embodiments, the sample preparation cartridge 500 may be configured to receive two needles, such as both a high-volume needle 900 and a low-volume needle 910.

[0110] AST cartridge embodiment: 12A and 12B show views of an AST cartridge 1200 in accordance with an embodiment of the present disclosure. The AST cartridge 1200 represents an exemplary embodiment of the AST cartridge 232. In particular, FIG. 12A shows the AST cartridge 1200 after assembly, while FIG. 12B shows an exploded view of the components within the AST cartridge 1200. The AST cartridge 1200 includes a cover 1202, a septum 1208, and a base 1212. In some embodiments, the cover 1202 is disposed over the septum 1208, which is disposed over the base 1212.

[0111] In some embodiments, the base 1212 includes a plurality of reaction wells 1214. In some embodiments, the number of reaction wells 1214 in the base 1212 may be in the range of about 50 to 200, such as, for example, 100 reaction wells. In some embodiments, each reaction well 1214 may hold a volume in the range of about 20 to 50 μL, such as, for example, 30 μL. Each reaction well 1214 of the plurality of reaction wells 1214 may contain a predetermined concentration of an antimicrobial agent for reacting with a respective aliquot of the enriched sample containing the pathogen. In some embodiments, the antimicrobial agent disposed in each reaction well 1214 may be in a liquid state or in a dried or lyophilized form. In some embodiments, the antimicrobial agent disposed in the reaction well 1214 may be added to each reaction well 1214 in the base 1212 by the first or second pipettor 202, 204 prior to dispensing the plurality of aliquots of the enriched sample into the reaction well 1214. In some embodiments, the antimicrobial agent disposed within each reaction well 1214 may be added to each reaction well 1214 during manufacture and / or assembly of the AST cartridge 1200.

[0112] In some embodiments, the first or second pipettor 202, 204 in the analyzer 200 may dispense multiple aliquots of the enriched sample into the reaction wells 1214 in the AST cartridge 1200 after concentration and enrichment of the pathogens in the sample in the processing tube 700. In some embodiments, each reaction well 1214 may be configured to receive an aliquot of the enriched sample containing the pathogens by non-contact dispensing with a needle (e.g., a needle 900 or 910 coupled to the first or second pipettor 202, 204). In some embodiments, the needle may pierce the septum 1208 of each reaction well 1214 to dispense each aliquot without contacting the bottom surface of each reaction well 1214. In some embodiments, the volume of each aliquot dispensed by the needle 900 or 910 may be in the range of about 0.5 μL to about 10 μL.

[0113] In some embodiments, each reaction well 1214 includes a conical shape and a bottom wall. In some embodiments, the bottom wall of each reaction well 1214 may have a diameter of less than about 2 mm. In some embodiments, non-contact dispensing of multiple aliquots into each reaction well 1214 may include the use of jet dispensing, in which a needle 900 or 910 in the sample preparation cartridge 500 pierces a septum of each of the reaction wells 1214 without contacting the bottom wall of each reaction well 1214.

[0114] In some embodiments, the bottom wall of each reaction well 1214 may be optically clear and configured for optical interrogation. In some embodiments, the bottom wall of each reaction well 1214 may be configured for optical interrogation, such as by the AST subsystem 250 in the analyzer 200. In some embodiments, the bottom wall of each reaction well 1214 may be configured for fluorescent microscopy, such as by the AST subsystem 250 in the analyzer 200. In some embodiments, the multiple reaction wells 1214 may be configured to fit into corresponding wells in a temperature control block in the AST subsystem 250, and the temperature control block may be configured to heat the sides of the multiple reaction wells 1214.

[0115] The septum 1208 seals each of the reaction wells 1214 of the base 1212. In some embodiments, the septum 1208 may be referred to as a sealing cap mat. In some embodiments, the septum 1208 may be comprised of a unitary structure that extends across the reaction wells 1214 of the base 1212. In some embodiments, the septum 1208 may include multiple parts assembled together, each part covering each of the reaction wells 1214 of the base 1212. The septum 1208 may be configured to receive a needle, such as the needle 900 or 910 coupled to the first or second pipettor 202, 204. In some embodiments, the needle may form a hole in the septum 1208 during insertion, and as a result of the material of the septum 1208, the hole in the septum 1208 may be blocked when the needle is removed. In some embodiments, the diaphragm 1208 may include at least one of rubber, polytetrafluoroethylene (PTFE), thermoplastic elastomer (TPE), silicone, butyl rubber, or combinations thereof. In some embodiments, the diaphragm 1208 may be constructed of a bilayer of polytetrafluoroethylene (PTFE) and another material selected from the group consisting of silicone, rubber, and butyl rubber. In some embodiments, the diaphragm 1208 may have a thickness in the range of about 1-2 mm.

[0116] In some embodiments, the cover 1202 includes a plurality of openings 1206, each opening 1206 aligning with a respective reaction well 1214 of the plurality of reaction wells 1214 in the base 1212. In some embodiments, the cover 1202 may also include an identifier 1210. The identifier 1210 may be an identifier of the AST cartridge 1200 that may be scanned by the analyzer 200 to perform antimicrobial susceptibility testing. In some embodiments, the identifier 1210 may be at least one of an identification code, a bar code, or a data matrix.

[0117] In some embodiments, the cover 1202 may fit over the septum 1208 and base 1212 to form the assembled AST cartridge 1200. In some embodiments, the septum 1208 is overmolded onto the cover 1202 to form a combined part, and the combined part is assembled onto the base 1212 by at least one of a snap fit connection or mechanical fasteners. In some embodiments, the cover 1202, septum 1208, and base 1212 may be interlocked or fastened together by one or more mechanical fasteners.

[0118] In some embodiments, the base 1212, septum 1208, and cover 1202 each include an opening 1204 in the center of the AST cartridge 1200. In some embodiments, the opening 1204 may be a circular hole that accommodates insertion by a pipette tip (e.g., first and second pipettors 202, 204) for moving the AST cartridge 1200 within the analyzer 200. In some embodiments, the openings 1204 in the base 1212, septum 1208, and cover 1202 may be aligned with one another upon assembly of the AST cartridge 1200.

[0119] In some embodiments, the base 1212 may be made of a polystyrene (PS) material. In some embodiments, the cover 1202 may be made of a polypropylene (PP) or polycarbonate (PC) material. In some embodiments, the dimensions of the assembled AST cartridge 1200 may be approximately 135 mm (length) x 35 mm (width) x 10 mm (height).

[0120] FIG 13 illustrates a low volume needle 910 being inserted into an AST cartridge 1200, according to an embodiment of the present disclosure. In particular, FIG 13 illustrates the needle shaft 914 of the needle 910 penetrating the septum 1208 of the AST cartridge 1200 into the reaction well 1214. In some embodiments, the needle shaft 914 may form an opening in the septum 1208 during insertion. The opening in the septum 1208 may close when the needle shaft 914 is removed as a result of the material of the septum 1208.

[0121] Embodiments of modules and subsystems in an analytical device: 14A and 14B show diagrams of example centrifuges used in the analytical device 200 according to embodiments of the present disclosure. In particular, FIG. 14A shows a first centrifuge 1402 that may be used to centrifuge a sample in a processing tube 1404, while FIG. 14B shows a second centrifuge 1412 that may be used to centrifuge an enriched sample in an AST cartridge 1414. The first and second centrifuges 1402, 1412 represent exemplary embodiments of the first and second centrifuges 240, 242, respectively, shown in FIG. 3B. The processing tube 1404 and the AST cartridge 1414 represent exemplary embodiments of the processing tube 700 and the AST cartridge 1200, respectively, shown in FIGS. 7A-7B and 12A-12B.

[0122] In some embodiments, the first centrifuge 1402 may be a high-speed centrifuge configured to apply a relative centrifugal acceleration (RCF) or g-force of about 12,000 G to the processing tube 1404. In some embodiments, the second centrifuge 1412 may be a low-speed centrifuge configured to apply a relative centrifugal acceleration (RCF) or g-force of about 3,000 G to the AST cartridge 1414.

[0123] In some embodiments, the first centrifuge 1402 may hold the processing tube 1404 in a first orientation and apply 45 degree swinging bucket centrifugation to the processing tube 1404. In some embodiments, the second centrifuge 1412 may hold the AST cartridge 1414 in a different orientation and apply 90 degree swinging bucket centrifugation such that the AST cartridge 1414 moves to a vertical position within the second centrifuge 1412.

[0124] In some embodiments, the first centrifuge 1402 and the second centrifuge 1412 may simultaneously centrifuge multiple processing tubes 1404 and AST cartridges 1414, respectively. For example, the first centrifuge 1402 may be configured to hold two processing tubes 1404 at the same time and centrifuge them together. In another example, the second centrifuge 1412 may be configured to hold two AST cartridges 1414 at the same time and centrifuge them together. In some embodiments, the processing tubes 1404 may be moved into the first centrifuge 1402 during the concentration and enrichment steps to obtain an enriched sample containing pathogens.

[0125] 15A, 15B, 15C, and 15D show diagrams of an example of an enrichment subsystem 1500 used in an analysis device 200 according to an embodiment of the present disclosure. The enrichment subsystem 1500 represents an embodiment of an example of the enrichment subsystem 246 shown in FIG. 3B. In some embodiments, the enrichment subsystem 1500 may apply a swinging motion to the processing tube 700 to allow vibration and mixing of the sample with other materials. In some embodiments, the processing tube 700 may be placed in the enrichment subsystem 1500 by a pipetting system (e.g., the first or second pipettor 202, 204) and pathogens in the processing tube 700 may be mixed with a growth medium to grow the pathogens and enrich the sample. In some embodiments, the processing tube 700 may be placed in the enrichment subsystem 1500 by a pipetting system (e.g., the first or second pipettor 202, 204) and one or more lysis reagents may be mixed with the blood sample in the processing tube 700 to lyse blood cells in the blood sample. In some embodiments, the mixing functionality of the enrichment subsystem 1500 may be used in sample processing for both pathogen identification and / or antimicrobial susceptibility testing in the analyzer 200.

[0126] In some embodiments, the enrichment subsystem 1500 may be a mixer that rotates one or more processing tubes 700 in a horizontal position by rocking the processing tubes 700 back and forth at an angle of ±30°. In some embodiments, four processing tubes 700 may be loaded into the enrichment subsystem 1500 at one time. In some embodiments, the enrichment subsystem 1500 may include a magnet 1501 that reciprocates between an up position and a down position. In some embodiments, one or more processing tubes 700 may include magnetic beads configured to attach to the concentrated pathogens in the one or more processing tubes 700. In some embodiments, the magnet 1501 may be used to retain the concentrated pathogens attached to the magnetic beads in the one or more processing tubes 700.

[0127] In some embodiments, the enrichment subsystem 1500 may include an additional or alternative independent magnet station used to hold the enriched pathogens attached to the magnetic beads in the processing tube 700 and / or the AST cartridge 1200. In some embodiments, the magnet station in the enrichment subsystem 1500 may be used to move the pathogens in each aliquot of the enriched sample (e.g., in the reaction wells 1214 of the AST cartridge 1200) to the bottom wall of each reaction well 1214 for acquiring an image of the AST cartridge 1200. In some embodiments, the pathogens in each aliquot may be attached to magnetic beads that allow for migration of the pathogens to the bottom wall of the reaction well 1214.

[0128] 16A, 16B, and 16C show diagrams of an example of a mechanical device 1600 used in the analytical device 200 according to an embodiment of the present disclosure. The mechanical device 1500 represents an embodiment of the example of the mechanical device 248 shown in FIG. 3B. In some embodiments, the mechanical device 1600 can hold two processing tubes 700 in a vertical position and provide a high-speed vibrational motion to the processing tubes 700, for example, to perform lysis of microorganisms in the sample. In some embodiments, the mechanical device 1600 can be an agitator. In additional or alternative embodiments, the mechanical device 1600 can include an ultrasonic generator configured to sonicate the processing tubes 700 to agitate the sample.

[0129] 17A and 17B show diagrams of an AST cartridge 1200 interfacing with an AST subsystem 1700 in an analyzer according to an embodiment of the present disclosure. The AST subsystem 1700 represents an exemplary embodiment of the AST subsystem 250 shown in FIG. 3B. In some embodiments, the AST subsystem 1700 may be an imaging subsystem configured to perform optical interrogation of the enriched sample in the AST cartridge 1200. In some embodiments, the AST subsystem 1700 may include a microscope 1702, a scanning stage 1706, and a thermal block 1708.

[0130] In some embodiments, the AST cartridge 1200 may be placed in the thermal block 1708 of the AST subsystem 1700 by a pipettor (e.g., the first or second pipettor 202, 204) in the analyzer 200. In some embodiments, the thermal block 1708 may surround all sides of the reaction wells 1214 of the AST cartridge 1200 when the AST cartridge 1200 is placed therein. In some embodiments, the thermal block 1708 may provide 37° C. temperature control for the AST cartridge 1200. In some embodiments, a heating lid 1710 may be placed on top of the AST cartridge 1200 when placed in the thermal block 1708 of the AST subsystem 1700. The microscope 1702 may be configured to obtain optical readings of the bottom walls of each reaction well 1214 in the AST cartridge 1200 via the thermal block 1708.

[0131] In some embodiments, the scanning stage 1706 may be comprised of a motorized XYZ translation stage that allows for motorized positioning of the microscope 1702 over the AST cartridge 1200. In some embodiments, the microscope 1702 may be configured to scan all reaction wells 1214 in the AST cartridge 1200 at one or more wavelengths (e.g., two wavelengths) in less than about 300 seconds. In some embodiments, the microscope 1702 may use a 10X objective lens for optical readout. In some embodiments, the microscope 1702 may use two optical channels to detect different fluorescent signals (e.g., green and red fluorescence). In some embodiments, the microscope 1702 may scan the AST cartridge using two different wavelengths, for example, a 490 nm wavelength for excitation and a 520 nm wavelength for emission (e.g., for detecting green fluorescence) and a 540 nm wavelength for excitation and a 620 nm wavelength for emission (e.g., for detecting red fluorescence).

[0132] In some embodiments, the microscope 1702 may be configured to perform fluorescent microscopy of the reaction wells 1214 in the AST cartridge 1200. In some embodiments, prior to acquiring one or more images using the microscope 1702, the first or second pipettor 202, 204 may dispense a first fluorescent dye and / or a second fluorescent dye into each reaction well 1214 in the AST cartridge 1200 to stain pathogens in the aliquot. In some embodiments, the first and / or second fluorescent dye may be delivered to the reaction wells 1214 after an aliquot of the enriched sample is incubated in the reaction wells 1214 of the AST cartridge 1200. In some embodiments, the first fluorescent dye may comprise a DNA-binding dye that labels live cells in the aliquot of reaction well 1214, while the second fluorescent dye may comprise a fluorescent intercalating agent that cannot cross intact cell membranes, and therefore labels only dead cells whose membranes are compromised, in the aliquot of reaction well 1214. For example, the first fluorescent dye may be SYBR® Green or another fluorescent dye that fluoresces green (e.g., with an emission wavelength of about 500-560 nm), and the second fluorescent dye may be propidium iodide (PI) or another fluorescent agent that fluoresces red (e.g., with an emission wavelength of about 560-700 nm).

[0133] In some embodiments, the first or second pipettor 202, 204 may use a jet dispensing method to perform non-contact dispensing in which the first or second needle 512 or 514 (coupled to the first or second pipettor 202, 204) penetrates the septum 1208 of each of the reaction wells 1214 and the first or second needle 512 or 514 does not contact the bottom wall of each reaction well 1214 to avoid cross-contamination between the reaction wells of the first or second fluorescent dye and / or the first or second fluorescent dye. In some embodiments, the amount of the first and / or second fluorescent dye dispensed by the first or second needle 512 or 514 into each reaction well 1214 may be in the range of about 0.5 microliters to 10 microliters. In some embodiments, the microscope 1702 can perform fluorescent microscopy for image acquisition of the AST cartridge 1200 by acquiring one or more fluorescent images to detect the first fluorescent dye and / or the second fluorescent dye in each reaction well 1214 of the AST cartridge 1200. In some embodiments, the microscope 1702 can be configured to detect one fluorescent dye, two fluorescent dyes, or the like, to analyze the fluorescent images of the AST cartridge 1200.

[0134] In some embodiments, the microscope 1702 may be coupled to a processor (e.g., processing unit 116) configured to perform image analysis and data processing of one or more images obtained from the AST cartridge 1200. In some embodiments, the processor coupled to the microscope 1702 may analyze one or more images obtained from the AST cartridge 1200 by calculating the number of fluorescent pathogens in each reaction well 1214 in the AST cartridge 1200. In some embodiments, the processor may determine whether the pathogens in the sample are resistant to various antimicrobial agents based on calculating the number of live and dead cells in the reaction wells 1214 of the AST cartridge 1200 and applying various rules. For example, the processor may identify whether a particular pathogen is resistant by determining whether the ratio of the number of dead cells (e.g., detected by red fluorescence in one or more images) to the number of live cells (e.g., detected by green fluorescence in one or more images) is below a predetermined threshold for a given antimicrobial concentration.

[0135] In another example, the processor may identify whether a particular pathogen is resistant by determining whether a ratio of the number of dead cells to the number of live cells for an aliquot of the enriched sample in the absence of an antimicrobial agent in the reaction well 1214 exceeds a predetermined threshold. In another example, the processor may identify whether a particular pathogen is resistant by determining whether a ratio of the number of live cells for an aliquot cultured with an antibiotic to the number of live cells for an aliquot cultured without an antibiotic exceeds a predetermined threshold. In yet another example, the processor may identify whether a particular pathogen is resistant by determining whether a ratio of the number of live cells for an aliquot cultured with an antibiotic to the number of live cells for the aliquot at time zero (e.g., before incubation / reaction) exceeds a predetermined threshold. In yet another example, the processor may identify whether a particular pathogen is resistant by determining whether a ratio of the brightness of live cells in one or more images of an aliquot cultured with an antibiotic exceeds a predetermined threshold relative to the ratio of the brightness of live cells in one or more images of an aliquot at time zero (e.g., before incubation / reaction). In some embodiments, the processor may apply one or more, or any combination, of these exemplary rules to identify highly resistant pathogens and determine whether a particular pathogen is resistant to a particular antimicrobial agent. In some embodiments, the microscope 1702 and processor may acquire and process images of the reaction wells 1214 in the AST cartridge 1200 in which multiple replicates of each reaction (e.g., between an aliquot of enriched sample and an antimicrobial agent) are performed.

[0136] In some embodiments, the processor may determine a minimum inhibitory concentration (MIC) of an antibiotic or antimicrobial agent that inhibits growth of a particular pathogen based on analyzing one or more images of the AST cartridge 1200. In some embodiments, different aliquots may be used to culture the pathogen with different antibiotic concentrations. In some embodiments, the processor may determine the MIC as the minimum concentration of antibiotic at which the reaction does not show significant growth relative to the initial number of pathogens. In some embodiments, the processor may determine the MIC by extrapolating the results of intermediate antimicrobial concentrations. In some embodiments, the processor may use the obtained value for the MIC to determine whether the pathogen is resistant, intermediate, or susceptible based on a lookup table or set of logic rules stored in a database (e.g., database 110).

[0137] Example of operation method: FIG. 18 illustrates a flow chart diagram of a method 1800 for performing AST of a sample, according to an embodiment of the present disclosure. In some embodiments, the method 1800 describes steps for performing AST using various components in an AST system, including the analyzer 108, 200, sample preparation cartridge 114, 224, 500, processing tube 113, 510, 700, AST cartridge 115, 232, 1200, controller 109, and processing unit 116, as previously described with reference to FIGS. 1-17. It should be understood that the operations shown in the method 1800 are not exhaustive and that other operations may be performed before, after, or between the operations shown. In various embodiments of the present disclosure, the operations of the method 1800 may be performed in different orders and / or may differ.

[0138] The method 1800 of Figure 18 begins at step 1802, where a sample preparation cartridge and a sample container are received at an analytical device. In some embodiments, the analytical device 200 may receive the sample preparation cartridge 224 and the sample container that are placed in the sample cartridge drawer 220 and the sample drawer 210, respectively, of the analytical device 200 by a user or operator of the analytical device 200. In some embodiments, the sample container includes a sample collected from a patient, the sample including a pathogen. In some embodiments, the sample in the sample container may include whole blood, urine, sterile bodily fluid, or other sample collected from a patient.

[0139] In step 1804, a first needle from the sample preparation cartridge is placed into the pipettor system of the analytical device. In some embodiments, the needle 512 or 514 from the sample preparation cartridge 500 is placed into the first or second pipettor 202, 204 by moving the pipette tip of the first or second pipettor 202, 204 up the sample preparation cartridge 500 and pressing against and engaging a plastic body portion of the needle (e.g., plastic body 902 or 912).

[0140] In step 1806, a first needle is inserted into a sample vessel using the pipettor system. In some embodiments, after attachment of the needle 512 or 514, the first or second pipettor 202, 204 may be moved above the sample vessel in the sample drawer 210 before pushing the needle 512 or 514 down to insert it into the sample vessel.

[0141] In step 1808, the sample from the sample container is transferred through the first needle to a processing tube in the sample preparation cartridge. In some embodiments, the needle 512 or 514 may draw the sample up into a plastic body reservoir of the first or second pipettor 202, 204, which then moves to the sample preparation cartridge 500 to transfer the sample to a processing tube 510 in the sample preparation cartridge 500.

[0142] To transfer a sample from a plastic body reservoir of the first or second pipettor 202, 204 to the processing tube 510, a needle 512 or 514 (coupled to the first or second pipettor 202, 204) may pierce a septum of the processing tube (e.g., septum 704 of processing tube 700). The first or second pipettor 202, 204 may then dispense the sample through the needle 512 or 514 into the processing tube.

[0143] In step 1810, the analytical device is used to concentrate and enrich the pathogens in the sample in a processing tube to obtain an enriched sample in the processing tube. In some embodiments, concentrating and enriching the sample in step 1810 may use components in the analytical device 200 to perform a series of steps including centrifugation, removal of fluid, addition of growth medium, and enumeration of pathogens in the enriched sample.

[0144] In step 1812, multiple aliquots of the enriched sample are dispensed into multiple reaction wells in an AST cartridge in the analyzer. In some embodiments, a first or second pipettor 202, 204 in the analyzer 200 may dispense multiple aliquots of the enriched sample into reaction wells 1214 in the AST cartridge 1200. In some embodiments, each aliquot corresponds to a respective reaction well 1214 in the AST cartridge 1200, and each reaction well 1214 may contain a predetermined concentration of an antimicrobial agent to react with a respective aliquot of the enriched sample containing the pathogen.

[0145] In step 1814, the aliquots in the reaction wells of the AST cartridge are incubated for a predetermined time to allow a reaction to occur between the pathogens in the aliquots in each reaction well and the antimicrobial agent. In some embodiments, the first pipettor 202 may move the AST cartridge 1200 to the AST subsystem 250 or 1700 and place the reaction wells 1214 in a thermal block or heater for temperature controlled incubation. In some embodiments, the predetermined period for incubating the aliquots in the reaction wells 1214 is about 2 hours or less.

[0146] In some embodiments, after incubating the aliquots in each reaction well 1214, the first pipettor 202 moves the AST cartridge 1200 to a centrifuge (e.g., centrifuge 242 or 1412) in the analyzer 200 and moves the pathogens in each aliquot to the bottom wall of each reaction well 1214 for acquiring an image of the AST cartridge 1200. In some embodiments, after incubating the aliquots in each reaction well 1214, the first pipettor 202 may move the AST cartridge 1200 to the enrichment subsystem 150, where a magnet (such as a magnet station) may be used to move the pathogens in each aliquot to the bottom wall of each reaction well 1214 and an image of the AST cartridge 1200 may be acquired. In some embodiments, the pathogens in each aliquot may be attached to magnetic beads.

[0147] In step 1816, an image of each reaction well in the AST cartridge is acquired using a microscope in the analyzer. In some embodiments, the first pipettor 202 moves the AST cartridge 1200 to the AST subsystem 1700, and the AST cartridge 1200 is imaged by the microscope 1702 using a scanning stage 1706. In some embodiments, each reaction well 1214 includes a bottom wall having an inner surface and an outer surface, and a reaction between the pathogen in the aliquot in each reaction well 1214 and the antimicrobial agent occurs above the inner surface of the bottom wall of each reaction well 1214 in the AST cartridge 1200. In some embodiments, an image of the AST cartridge 1200 is acquired by the microscope 1702 at the outer surface of the bottom wall of each reaction well 1214 of the AST cartridge 1200. In some embodiments, the microscope 1702 is configured to acquire one or more images taken from below the outer surface of the AST cartridge 1200 outer surface. In some embodiments, the microscope 1702 may be configured to acquire one or more fluorescent images to detect the first fluorescent dye and / or the second fluorescent dye in each reaction well 1214 of the AST cartridge 1200.

[0148] In step 1818, the susceptibility of the pathogens in each reaction well to the antimicrobial agent is determined by analyzing the images. In some embodiments, a processor (e.g., processing unit 116) coupled to the microscope 1702 in the analysis device 200 may be used to determine the susceptibility of the pathogens in each reaction well 1214 to the antimicrobial agent by analyzing one or more images obtained from the microscope 1702. In some embodiments, the processor may analyze the one or more images by calculating the number of fluorescent pathogens in each reaction well 1212 in the AST cartridge 1200. In some embodiments, the processor may determine the susceptibility of the pathogens by determining whether one or more of the pathogens are susceptible, intermediate, or resistant to the antimicrobial agent in each reaction well 1214 based on staining of the cells in the aliquot with a first fluorescent dye.

[0149] In some embodiments, the processor may determine the susceptibility of the pathogens by determining whether one or more of the pathogens in each reaction well 1214 are susceptible, intermediate, or resistant to the antimicrobial agent based on staining of the cells in the aliquots with the first fluorescent dye and / or the second fluorescent dye. In some embodiments, the processor may determine that a particular pathogen is resistant to a particular antimicrobial agent in the reaction well 1214 by determining that a ratio of the number of dead cells to the number of live cells is below a predetermined threshold for a given concentration of the particular antimicrobial agent based on staining of the cells in the aliquots with the first fluorescent dye and / or the second fluorescent dye. In some embodiments, the processor may determine that the ratio of the number of dead cells to the number of live cells is below a predetermined threshold of about 20% or about 10%.

[0150] In some embodiments, a processor coupled to the microscope 1702 may be configured to detect highly resistant pathogens based on an accelerated response in one or more reaction wells 1214 resulting from the use of a high dose of antimicrobial agent in the reaction wells 1214. In particular, a first reaction well 1214 of the plurality of reaction wells 1214 may include a first concentration of antimicrobial agent, the first concentration being a high dose that is significantly higher than a clinical breakpoint for the antimicrobial agent and the pathogen. In some embodiments, the clinical breakpoint may represent a concentration or dose of antimicrobial agent or antibiotic used to define whether an infection by a particular pathogen is susceptible to that antimicrobial agent or antibiotic and may be successfully treated. In some embodiments, using an antimicrobial agent concentration higher than the clinical breakpoint may accelerate a reaction between the antimicrobial agent and the concentrated pathogen in the aliquot in the reaction well. In some embodiments, using a higher antimicrobial agent concentration may cause the processor to determine susceptibility of the pathogen by comparing an image of the first reaction well to a control to determine a highly resistant pathogen within about an hour.

[0151] In addition to determining the susceptibility of the pathogen, the processor may be further configured to determine a minimum inhibitory concentration (MIC) of a particular antimicrobial agent for inhibiting the growth of the particular pathogen in one or more pathogens based on the image of the AST cartridge 1200. Furthermore, the processor may be configured to determine whether a particular pathogen is susceptible, intermediate, or resistant to a particular antimicrobial agent by analyzing a set of rules in a database (e.g., database 110) communicatively coupled to the processor. In some embodiments, the one or more databases 110 may be configured to store pathogen classification data and / or results of a previous pathogen identification workflow (e.g., performed by the analyzer 200). In some embodiments, the processor may retrieve the pathogen classification data and / or results from the one or more databases 110 and use the retrieved data to determine one or more rules to apply to make a determination of resistant / intermediate / susceptible from the MIC.

[0152] 19 illustrates a flow chart diagram of a method 1900 for manufacturing or assembling an AST cartridge, according to an embodiment of the present disclosure. In some embodiments, the method 1900 may describe the manufacturing and / or assembly of an AST cartridge, such as the AST cartridges 232, 1200 described above with reference to FIGS. 3B-17. It should be understood that the operations illustrated in the method 1900 are not exhaustive and that other operations may be performed before, after, or between the illustrated operations. In various embodiments of the present disclosure, the operations of the method 1900 may be performed in a different order and / or may be modified.

[0153] The method 1900 of Figure 19 begins with step 1902, in which a cover with a plurality of openings is manufactured. In some embodiments, the cover 1202 of the AST cartridge 1200 with the openings 1206 can be manufactured using injection molding. In some embodiments, the cover 1202 can be manufactured from polypropylene (PP), polycarbonate (PC), or another plastic material.

[0154] In step 1904, a diaphragm is overmolded onto the cover such that a first side of the diaphragm traverses the multiple openings in the cover. In some embodiments, the cover 1202 may be a substrate onto which the diaphragm 1208 is molded directly into a single, integral piece. In some embodiments, the diaphragm 1208 may be made from a bilayer of polytetrafluoroethylene (PTFE) and another material selected from the group consisting of silicone, rubber, and butyl rubber. In some embodiments, the diaphragm 1208 may be made from at least one of rubber, polytetrafluoroethylene (PTFE), thermoplastic elastomer (TPE), silicone, butyl rubber, or a combination thereof.

[0155] In step 1906, a base including a plurality of reaction wells may be manufactured. In some embodiments, the base 1212 may be manufactured using injection molding. In some embodiments, the base 1212 may be manufactured from polystyrene (PS). In some embodiments, manufacturing the base 1212 includes manufacturing a plurality of reaction wells 1214 connected together as a single component. In some embodiments, the plurality of reaction wells 1214 in the base 1212 may be manufactured in a conical shape. In some embodiments, a diameter of a bottom wall of each reaction well 1214 may be less than about 2 millimeters.

[0156] In some embodiments, during manufacture of the base 1212, a predetermined concentration of antimicrobial agent may be added in liquid form to each reaction well 1214 before the base 1212 is attached to the septum 1208. In some embodiments, the antimicrobial agent in each reaction well 1214 may be dried to obtain a dried or lyophilized form of the antimicrobial agent using forced air. In some embodiments, the steps of adding and drying the antimicrobial agent in each reaction well 1214 may be completed within a predetermined time to prevent degradation of the antimicrobial agent. In some embodiments, the predetermined time of adding and drying the antimicrobial agent is about 10 minutes, 15 minutes, etc.

[0157] In step 1908, the base may be attached to the second side of the septum such that the second side of the septum seals across the plurality of reaction wells in the base. In some embodiments, the first side of the septum 1208 is overmolded to the cover 1202 and the second side of the septum 1208 is coupled to the base 1212 to provide a sealing mechanism over the reaction wells 1214 in the base 1212. In some embodiments, the base 1212 may be coupled to the second side of the septum 1208 using at least one of a snap fit coupling or a mechanical fastener. In some embodiments, when the base 1212 is attached to the second side of the septum 1208, each opening 1206 of the cover 1202 may be aligned with a respective reaction well 1214 of the plurality of reaction wells 1214 in the base 1212.

[0158] Usage example In some embodiments, the AST system described above can be used to determine the minimum inhibitory concentration in antibiotic susceptibility testing of blood samples. In some embodiments, the blood sample is added to a reaction tube containing a lysing agent. In some embodiments, the amount of sample can depend on the size of the reaction tube used. For example, if the reaction tube has a volume of 15 mL, the sample volume in the tube will be less than 15 mL, for example 10 mL. In some embodiments, an additional reagent such as a protease may be added to the reaction tube to improve the lysing action. For example, 100 μL to 2 mL of protease may be added. To lyse and concentrate the sample, the sample is incubated at low speed and then centrifuged. To prepare the matrix for the enrichment step, the sample supernatant may be discarded until a desired amount of sample remains. In some embodiments, a volume in the range of 200 μL to 2 mL is maintained after concentration. In some embodiments, a volume of about 500 μL is maintained after concentration.

[0159] In some embodiments, an enrichment step is optionally performed. Prior to such enrichment, culture medium broth may be added and vortexed. Enrichment may be performed at enrichment temperature with rotary mixing for a preferred period of time. In some embodiments, the total volume of enrichment may be determined by the volume of culture medium added relative to the volume of enriched sample. In some embodiments, the enrichment volume is about 5 mL (e.g., 500 μL of enriched sample + 4500 μL of culture medium). Those skilled in the art will recognize that other volumes and ratios may be used without departing from the present invention.

[0160] In some embodiments, immunomagnetic separation may be used to separate the bacteria of interest from the blood in the sample. For example, magnetic beads bound to biotinylated molecules associated with the bacteria of interest may be used. These coated magnetic beads may be added to the enriched sample in a reaction tube and incubated. In some embodiments, non-specific magnetic beads may be used to capture any bacteria present in the sample.

[0161] In some embodiments, after capture of the bacteria by the magnetic beads, a set of reagents may be used to digest, reduce and / or remove debris from the blood sample. As an example, a protease in the range of 2 μL to 200 μL may be used for this purpose, and / or DNAses to digest any free DNA may be used.

[0162] After incubation, collection of the magnetic beads can be performed by placing the reaction tube on or near a magnet. Once the beads have collected in the part of the reaction tube closest to the magnet, the supernatant is removed and discarded with a pipette. The remaining beads containing the sample bacteria can be washed according to a washing process. Once washing is complete, the beads can be resuspended in culture media. A desired amount of bead sample (e.g., 500 μL) can be extracted from the tube to a sample plate (e.g., a well plate), diluted as necessary, and vortexed to obtain a desired volume and concentration of sample for incubation and dispensed into multiple reaction wells of the plate. The sample plate can then be centrifuged to collect the bacteria at the bottom of the wells for imaging. In some embodiments, the sample plate is centrifuged at 3000 G for 1 minute.

[0163] The samples on the sample plate are then incubated for the desired time. For example, the samples are incubated for 5 hours. The sample plate is then centrifuged to collect the bacteria at the bottom of the wells for imaging. The incubated samples are then imaged from the bottom of the sample plate to measure the growth (and antimicrobial susceptibility only) of the samples.

[0164] Experimental Example: As described below, several experiments were performed to test various embodiments of the antibiotic susceptibility testing systems and methods described above.

[0165] Example 1: Determination of minimum inhibitory concentration of samples from blood collection tube samples This example describes a method for performing antibiotic susceptibility testing from blood collection tube samples according to embodiments disclosed herein.

[0166] First, streptavidin-coated beads were washed and immobilized. The beads with bound biotinylated molecules (in this case antibodies) were incubated overnight at room temperature with gentle rotation (25 rpm) in phosphate buffered saline (adjusted to pH 7.4). The typical binding capacity (100 μL) per mg of magnetic beads used is approximately 20 μg of biotinylated antibody. After coating, they were washed several times with phosphate buffered saline (adjusted to pH 7.4) containing 0.01% BSA (w / v). Finally, the beads were resuspended to the concentration required for the application. The antibody used was a biotinylated Klebsiella polyclonal antibody (PA1-73177, Invitrogen) and the magnetic beads used were Dynabeads MyOne Streptavidin T1 (65601, Thermofisher Scientific).

[0167] Blood samples were prepared from a single large pool of blood obtained from a 10 mL EDTA blood tube drawn from a blood donor and spiked with an appropriate amount of bacteria (Klebsiella pneumoniae ATCC13883) depending on the target inoculum (approximately 100 CFU / 10 mL). After the pool was spiked with bacteria, 10 mL was transferred to a pre-labeled 15 mL tube and lysis agent (Isolator BC0507C, 700 μL from Oxoid) and immiscible fluorocarbon oil (Fluorinert™ FC-40 F9755, 20 μL, Sigma-Aldrich) were added. For lysis and concentration, samples were briefly incubated (30 s) while spinning at low speed (10 rpm) and centrifuged at 12,000 g force for 5 min in a fixed angle rotor. The supernatant of each sample was discarded until 500 μL was used to prepare the matrix for the enrichment step. At this point, half of the samples were discontinued and 500 μL of these were plated on agar plates as a checkpoint to see how far the enrichment process had progressed and at what CFU the next step would begin.

[0168] For samples that were continued in the experiment, 4500 mL of media (Mueller-Hinton Broth) was added and vortexed for 1 min prior to the enrichment step, which was performed at 37°C with slow (10 rpm) rotation for 30 min. Once this step was completed, banks of dilutions were made and plated on agar media to capture the bacterial populations in each sample, growth times, and numbers of bacteria available for the immunomagnetic separation process.

[0169] To immunomagnetically separate bacteria from blood, 100 μL of antibody-coated beads were added to each sample tube. The incubation conditions used were 30 min at 37 °C with gentle rotation (25 rpm). After incubation, the bank dilutions with 100 μL of matrix were applied to LB agar plates to capture the number of bacteria (bound and unbound to beads). Collection of magnetic beads was performed by placing the tubes on a magnet for 15 min. After this time, the supernatant was pipetted off and discarded, and two washing steps were performed to clean the samples. For each washing step, 5 mL of washing buffer (phosphate-buffered saline, pH 7.4) was added, mixed gently with rotation (10 rpm) for 1 min at 37 °C, the tube was briefly placed on a magnet (1 min) to collect the beads, and the supernatant was pipetted and discarded. Once washing was complete, the beads were resuspended in 500 μL of incubation medium (Mueller Hinton cation adjusted broth) and vortexed for 10–20 seconds. To measure the amount of bacteria captured by the magnetic beads, 100 μL of the final matrix was used to create a bank of dilutions and spread onto LB agar plates incubated at 37°C for 16–18 hours. These samples were also observed under an inverted fluorescent microscope to identify the bacteria present in the samples and calculate the amount required for AST testing, which was the next and final step of this end-to-end assay. Five μL of each sample was transferred to a 384-well plate with a flat polystyrene film bottom and incubated with fluorescent dyes (SYBR Green and propidium iodide) for 10 minutes at room temperature in the dark. The 384-well plates were centrifuged at 3000 g for 1 minute in a swinging bucket setup and observed under an inverted fluorescent microscope. For each sample, a composite image was created covering the bottom of each well.

[0170] Based on this measurement, the sample was diluted (Mueller-Hinton Cation Adjusted Broth) to obtain a bacterial count of approximately 4x10 4 Bacterial samples were incubated at 37°C for 5 h in a final volume of 10 μL with different ciprofloxacin concentrations at a final concentration of 2 × 10 4CFU / mL. Ciprofloxacin concentrations tested were 0.125, 0.06, 0.03, 0.015, 0.008, and 0.004 mg / L. Three replicates were performed for each antibiotic concentration.

[0171] Samples were photographed after 0 min and 5 h incubation (with and without antibiotics). 5 μL of each sample was transferred to a 384-well plate with a flat polystyrene film bottom and incubated with fluorescent dyes (SYBR Green and propidium iodide) for 10 min at room temperature in the dark. The 384-well plates were centrifuged at 3000 g force for 1 min in a swinging bucket centrifuge and viewed under an inverted fluorescence microscope. Composite images were generated for each sample. Images were analyzed with custom software that counted bacteria, detected filamentous bacteria, counted red bacteria, and recorded bacterial brightness. These images are shown in Figure 21A.

[0172] The data obtained from the images was used to generate a graph showing that growth was observed at ciprofloxacin concentrations of 0.008 and 0.004 mg / L, but not at concentrations of 0.015 mg / L or greater (Figure 21B). These results, combined with bacterial counts and data analysis, indicate that the MIC for this strain is 0.015 mg / L, which is essentially consistent with the broth microdilution method, which gives an MIC of 0.03 mg / L for this strain. This graph is shown in Figure 21B.

[0173] Example 2: Determination of the minimum inhibitory concentration of samples from blood culture bottles This example describes how to perform antibiotic susceptibility testing starting from a blood culture bottle, according to embodiments disclosed herein.

[0174] First, streptavidin-coated beads were washed and immobilized. The washing buffer and immobilization process may vary depending on the application. The beads bound to the biotinylated molecules (in this case antibodies) were incubated overnight in phosphate-buffered saline (adjusted to pH 7.4) with gentle rotation (25 rpm) at room temperature. The typical binding capacity per mg of magnetic beads (100 μL) used is about 20 μg of biotinylated antibody. After coating, several washes were performed in phosphate-buffered saline (adjusted to pH 7.4) containing 0.01% BSA (w / v), after which the beads were finally resuspended to the concentration required for the application. The antibody used was a biotinylated Klebsiella polyclonal antibody (PA1-73177, Invitrogen) and the magnetic beads used were Dynabeads MyOne Streptavidin T1 (65601, ThermosFisher Scientific).

[0175] Blood samples were prepared from a single large pool of premixed blood to which an appropriate amount of bacteria (Klebsiella pneumoniae ATCC13883) was added depending on the target inoculum (approximately 100 CFU). After the pool was added with bacteria, 10 mL was transferred to a prelabeled blood culture system (442023, BD BACTEC™ Plus Aerobic / F) and mixed with a stirring device (150 rpm for 5 min). After the time had elapsed, a moderate amount of time (30 s to 1 min) was waited for the resin to settle. A 5 mL sample was then extracted.

[0176] Immunomagnetic separation was performed using 15 mL tubes containing 5 mL of matrix extracted from blood culture bottles and 100 μL of beads coated with anti-Klebsiella pneumoniae antibodies. The incubation conditions were 37 °C for 30 min with gentle rotation (25 rpm). After incubation, a bank of dilutions was made and applied with 100 μL of matrix to determine the number of bacteria present in each sample (bound and unbound to magnetic beads). Recovery of the magnetic beads was performed by placing the tubes on a magnet for 7.5 min. After this time, the supernatant was pipetted off and discarded, and three washing steps were performed to wash the samples. Each washing step involved adding 5 mL of medium (Mueller Hinton cation adjusted broth), vortexing for 1 min, mixing with gentle rotation (10 rpm) for 2 min at 37 °C, briefly placing the tubes on a magnet (2 min) to collect the beads, and pipetting and discarding the supernatant. After washing, the beads were resuspended in 500 μL of incubation medium (Mueller Hinton cation-adjusted broth) and vortexed for 1 min. To measure the amount of bacteria captured by the antibody-coated magnetic beads, 100 μL of the final matrix was used to make a dilution series and spread onto LB agar plates incubated at 37 °C for 16–18 h. Samples were also observed under an inverted fluorescence microscope to determine the number of bacteria in the samples and to calculate the adjustment required for AST testing. Five μL of each sample was transferred to a 384-well plate with a flat polystyrene membrane bottom and incubated with fluorescent dyes (SYBR Green and propidium iodide) for 10 min at room temperature in the dark. The 384-well plates were centrifuged at 3000 g for 1 min in a swinging bucket centrifuge and observed under an inverted fluorescence microscope. For each sample, a composite image was made covering the bottom of each well. The images were analyzed with custom software to determine the number of bacteria.

[0177] Based on this measurement, the sample was diluted (Mueller-Hinton Cation Adjusted Broth) to obtain a bacterial count of approximately 4x10 4 CFU / mL. Bacterial samples were cultured at a final concentration of 2x10 4Different gentamicin concentrations, measured in CFU / mL, were incubated in a final volume of 10 μL for 5 h at 37° C. Gentamicin concentrations were tested at 2, 1, 0.5, 0.25, 0.125, and 0.06 mg / L. Three replicates were performed for each antibiotic concentration.

[0178] Samples were analyzed after 0 and 5 hours of incubation (with and without antibiotics). 5 μL of each sample was transferred to a 384-well plate with a flat polystyrene film bottom and incubated with fluorescent dyes (SYBR Green and propidium iodide) for 10 minutes in the dark at room temperature. The 384-well plates were centrifuged at 3000 g force for 1 minute in a swinging bucket centrifuge and observed under an inverted fluorescence microscope. Composite images were generated for each sample. Images were analyzed with custom software that counted bacteria, detected filamentous fungi, counted red bacteria, and recorded bacterial brightness. These images are shown in Figure 22A.

[0179] Using the data obtained from the images, a graph was generated showing that growth was observed at 0.125 and 0.06 mg / L gentamicin, but no growth at concentrations above 0.25 mg / L (Figure 22B). These results, along with bacterial counts and data analysis, indicate that the MIC for this strain is 0.25 mg / L, and by broth microdilution, the MIC for this strain is 0.5-0.25 mg / L. This graph is shown in Figure 22B.

[0180] Example 3: Determination of the minimum inhibitory concentration of gentamicin for gram-negative bacteria The minimum inhibitory concentration was determined using embodiments disclosed herein for Klebsiella pneumoniae (ATCC13883), a strain of Gram-negative bacteria that is sensitive to gentamicin and has a minimum inhibitory concentration of 0.25-0.5 mg / mL. The bacteria were grown in liquid culture and grown to exponential growth phase in the culture medium. The culture was diluted with culture medium (Mueller-Hinton Cation Adjusted Broth) and incubated at 37°C for 5 hours at different concentrations of gentamicin, with a final concentration of 2x10 in a final volume of 10 μL. 4 CFU / mL. Gentamicin concentrations tested were 4, 2, 1, 0.5, 0.25, and 0.125 mg / L. Three replicates were performed for each antibiotic concentration.

[0181] Samples were analyzed after 0 and 5 hours of incubation (with and without antibiotics). Samples were incubated with fluorescent dyes (SYBR Green and propidium iodide) for 10 minutes at room temperature in the dark. After dye incubation, 1 μL of sample was diluted with 4 μL of buffer (0.9% NaCl) and transferred to a 384-well plate with a flat polystyrene film bottom. The 384-well plate was centrifuged at 3000 g for 1 minute in a swinging bucket and observed under an inverted fluorescence microscope. For each sample, a composite image was generated covering the bottom of each well. Images were analyzed with custom software that counted bacteria, detected filamentous fungi, counted red bacteria, and recorded bacterial brightness. These images are shown in Figure 23A.

[0182] The data obtained from the images was used to generate a graph showing that the bacteria grew at 0.125 mg / L gentamicin but not at concentrations of 0.25 mg / L or higher (Figure 23B). These results, combined with bacterial counts and data analysis, indicate that the MIC for this strain is 0.25 mg / L, which is consistent with the MIC obtained by the broth microdilution method. This result indicates that the MIC of gentamicin for these Gram-negative strains can be determined by using bacterial growth and bacterial characteristics after 5 hours of incubation at different antibiotic concentrations. This graph is shown in Figure 23B.

[0183] Example 4: Determination of the minimum inhibitory concentration of meropenem for Gram-negative bacteria The minimum inhibitory concentration for meropenem was determined for the Gram-negative strain Klebsiella pneumoniae (IHMA1977064) according to the broth microdilution method using embodiments disclosed herein. Bacteria were grown in liquid culture to exponential growth phase in culture medium. Cultures were diluted in culture medium (Mueller-Hinton Cation-Adjusted Broth) and incubated with different concentrations of meropenem for 5 h at 37° C. in a final volume of 10 μL at a final concentration of 2×10 4 Cultures were grown at CFU / mL. Meropenem concentrations were 32, 16, 8, 4, 2, and 1 mg / L of gentamicin. Three replicates were performed for each antibiotic concentration.

[0184] Samples were analyzed after 0 and 5 hours of incubation (with and without antibiotics). 5 μL of each sample was transferred to a 384-well plate with a flat polystyrene film bottom and incubated with fluorescent dyes (SYBR Green and propidium iodide) for 10 minutes in the dark at room temperature. The 384-well plates were centrifuged at 3000 g for 1 minute in a swinging bucket centrifuge and viewed under an inverted fluorescence microscope. For each sample, a composite image was created covering the bottom of each well. Images were analyzed with custom software that counted bacteria, detected filamentous fungi, counted red bacteria, and recorded bacterial brightness. These images are shown in Figure 24A.

[0185] Using the data obtained from the images, a graph was generated showing bacterial growth at meropenem concentrations of 2 and 1 mg / L, but no growth at concentrations of 4 mg / L and above (Figure 24B). These results, combined with bacterial counts and data analysis, determined that the MIC for this strain was 4 mg / L, and the broth microdilution method gave an MIC of 8 mg / L for this strain, achieving essential agreement. This graph is shown in Figure 24B.

[0186] Results showed that the MICs of these Gram-negative strains against meropenem could be determined by incubation with different antibiotic concentrations for 5 h and using bacterial growth and bacterial characteristics.

[0187] Example 5: Determination of the minimum inhibitory concentration of erythromycin against gram-positive bacteria According to the embodiments described herein, the minimum inhibitory concentrations of two strains of the Gram-positive bacteria Staphylococcus aureus, one susceptible (ATCC29213) and one resistant (ATCC43300) to erythromycin, were measured. The MICs of these strains were measured by broth microdilution method, with the MICs of erythromycin-susceptible Staphylococcus aureus ranging from 0.25 to 0.5 mg / L and the MICs of the resistant strains being 512 mg / L or higher. The bacterial strains were grown to the vegetative phase in liquid culture. The bacterial cultures were diluted in culture medium (Mueller-Hinton Cation Adjusted Broth) and cultured in medium containing different concentrations of gentamicin in a final volume of 10 μL to a final concentration of 2x10 4 CFU / mL and incubated for 5 hours at 37°C. Erythromycin-susceptible S. aureus strains were tested at 1, 0.5, 0.25, 0.125, 0.06, and 0.03 mg / L, and resistant strains were tested at 4, 2, 1, 0.5, 0.25, and 0.125 mg / L erythromycin. Three replicates were performed for each antibiotic concentration.

[0188] Samples were analyzed after 0 and 5 hours of incubation (with and without antibiotics). Samples were incubated with fluorescent dyes (SYBR Green and propidium iodide) for 10 min at room temperature in the dark. After dye incubation, 1 μL of sample was diluted with 4 μL of buffer (0.9% NaCl) and transferred to a 384-well plate with a flat polystyrene film bottom. The 384-well plate was centrifuged at 3000 g for 1 min in a swinging bucket system and observed under an inverted fluorescence microscope. For each sample, a composite image was created covering the bottom of each well. Images were analyzed with custom software that counted bacteria, detected filamentous fungi, counted red bacteria, and recorded bacterial brightness.

[0189] The data obtained from the images was used to generate graphs of erythromycin-susceptible and -resistant S. aureus (Figures 25A and 25B, respectively). S. aureus susceptible to erythromycin showed growth at 0.125 mg / L erythromycin, but not at concentrations above 0.25 mg / L (Figure 25A). These results, combined with bacterial counts and data analysis, indicated that the MIC for this strain was 0.25 mg / L, which is consistent with the MIC obtained by the broth microdilution method. Data analysis of the erythromycin-resistant S. aureus strain (Figure 25B) showed growth at erythromycin concentrations of 4 mg / L and below, indicating that the MIC for this strain was 4 mg / L or higher. The MIC obtained by the broth microdilution method was ≥ 512 mg / L, indicating that the strain was resistant to erythromycin by both methods (EUCAST and CLSI breakpoints indicate that S. aureus strains with an erythromycin MIC ≥ 2 are resistant). The results demonstrated that the erythromycin MIC of these Gram-positive strains can be determined by incubation with different antibiotic concentrations for 5 hours and using the bacterial growth and characteristics.

[0190] Example 6: Rapid detection of ampicillin-resistant bacteria To rapidly detect bacteria highly resistant to a particular antimicrobial agent, bacteria were grown for a short period (1 h) in the presence of a high dose of the antimicrobial agent. Two strains of Escherichia coli (E. coli), one susceptible (ATCC25922) and one resistant (ATCC700891) to ampicillin, were grown to exponential phase in culture media. The bacterial cultures were diluted with medium (Mueller-Hinton Cation-Adjusted Broth) to final concentrations of 128 mg / L, 64 mg / L, and 32 mg / L ampicillin, and incubated at 37°C for 1 h at a final concentration of 2x10 4 Cultures were counted at CFU / mL. Each antibiotic concentration was tested in triplicate.

[0191] Samples were visualized at time 0, representing bacteria before incubation and bacteria after 1 h incubation. 5 μL of each sample was transferred to a 384-well plate with a flat polystyrene film bottom and incubated with fluorescent dyes (SYBR Green and propidium iodide) for 10 min at room temperature in the dark. The 384-well plates were centrifuged at 3000 g for 1 min in a swinging bucket format and viewed under an inverted fluorescence microscope. For each sample, a composite image was created covering the bottom of each well. Images were analyzed with custom software that counted bacteria, detected filamentous fungi, counted red bacteria, and recorded bacterial brightness. These images are shown in Figure 26A.

[0192] Comparing the images of both E. coli strains at different concentrations of ampicillin, the ampicillin-susceptible E. coli strain with a broth microdilution MIC of 4 mg / L did not grow at any of the tested concentrations of ampicillin (Figure 26B), the bacteria had a filamentous morphology at the three tested concentrations, and red bacteria were also present at 128 mg / L ampicillin, indicating bacterial death, whereas the ampicillin-resistant E. coli strain with a broth microdilution MIC of ≥ 128 mg / L showed growth without the presence of filamentous or red bacteria after 1 hour at all three concentrations of ampicillin (Figure 26B).

[0193] The results indicate that ampicillin-resistant bacteria can be detected by incubating the bacteria with a high antibiotic concentration for one hour and using the bacterial growth and characteristics.

[0194] Example 7: Cleanup of blood samples using specific beads Different time conditions were evaluated to establish the optimal duration of bacterial capture by magnetic beads. First, streptavidin-coated beads were washed with a washing buffer based on the application. After washing, the beads were immobilized. The beads were coupled to a biotinylated molecule, such as an antibody as in this case, and incubated overnight at room temperature with gentle rotation (25 rpm) in phosphate-buffered saline (adjusted to pH 7.4). The binding capacity per mg (100 μL) of tested magnetic beads was typically around 20 μg of biotinylated antibody. After coating was completed, they were washed several times with phosphate-buffered saline (adjusted to pH 7.4) containing 0.01% BSA (w / v) and finally the beads were resuspended to the concentration required for the application. The antibody used was a biotinylated Klebsiella polyclonal antibody (PA1-73177, Invitrogen) and the magnetic beads used were Dynabeads MyOne Streptavidin T1 (65601, ThermosFisher Scientific).

[0195] Different capture times were evaluated on blood samples. Blood samples were prepared from a single large pre-shaken blood pool to which an appropriate amount of bacteria (Klebsiella pneumoniae ATCC13883) was added depending on the target inoculum. After the pool was added, 10 mL was transferred to a pre-labeled blood culture system (442023, BD BACTEC™, Plus Aerobic / F) and mixed with a stirring device (180 rpm for 5 min). Once the time had elapsed, an appropriate time was waited (30 s to 1 min) for the resin to settle, and then a 5 mL sample was extracted. No more than 10 mL of sample was extracted from each blood culture system.

[0196] 5 mL of blood obtained from the blood culture system was transferred to a 15 mL tube and the required amount of antibody-coated beads (100 μL) was added. The tubes were incubated at 37 °C for 30 min with gentle rotational motion (25 rpm). After incubation, 100 μL of matrix was used to create a dilution bank and dispensed into the plate. The tubes were placed on the magnet for a given time (15-7.5 min), the beads were collected, and the supernatant was discarded by pipetting. At this point, 5 mL of wash buffer was added and three corresponding wash batches were initiated. For each wash step, vortexing was performed for 1 min, the sample was incubated at 37 °C for 2 min with gentle rotational motion (25 rpm), the tube was placed on the magnet for a short time (5-2.5 min) to collect the beads, and the supernatant was discarded by pipetting. After washing, the beads were resuspended in 500 μL of culture medium (Mueller-Hinton cation-adjusted broth) and vortexed for 1 min. In the next step, 100 μL of matrix was used to create a dilution bank, which was then applied to a plate. In parallel, these samples were observed under a microscope to check the concordance of the two measurements (plate and microscope). To observe the samples under an inverted fluorescence microscope, 5 μL of each sample was transferred to a 384-well plate containing fluorescent dyes (SYBR-Green + propidium iodide). The plate was then incubated for 10 min at room temperature and centrifuged (3000 g force, 1 min) before visualization. Table 1 shows that the results were largely independent of the capture time.

[0197] [Table 1]

[0198] Example 8: Blood sample cleanup process using non-specific beads A series of reagents were evaluated to improve sample cleanup from cell debris derived from blood cell lysates. Blood samples were prepared from a single large volume of pre-shaken blood (150 rpm for 5 min). After shaking, 10 mL was transferred to a pre-labeled blood culture system (442023, BD BACTEC™ Plus Aerobic / F) and mixed on a stirring platform (180 rpm for 5 min). Once the time had elapsed, an appropriate time was waited (30 s to 1 min) for the resin to settle, and then a 10 mL sample was extracted. No more than 10 mL of sample was extracted from each blood culture system.

[0199] 10 mL blood samples drawn from the blood culture system were transferred to 15 mL tubes and lysed with variable amounts of lysis agent (Saponin S4521 at 2,8 g / 100 mL, 700 μL, Sigma-Aldrich). Immiscible fluorocarbon oil (Fluorinert™, FC-40 F9755, 20 μL, Sigma-Aldrich) was also added to the tubes used for the lysis and concentration steps. In addition to the lysis agent, other reagents such as protease were added to enhance the action (Protease from Aspergillus oryzae P6110, Sigma-Aldrich, 250 μL). The lysis and concentration steps required a short (30 s) spinning incubation of the samples at low speed (10 rpm), followed by centrifugation at 12000 g force for 5 min, discarding the supernatant to 500 μL, and preparing the matrix for IMS with 500 μL of TTGB 2X buffer. Once the initial matrix was obtained, complementary reagents such as DNAses (different concentrations and times) were added to improve removal of cell debris. Before adding the magnetic beads, the appropriate amount of bacteria (E. coli ATCC25922) was added to the samples depending on the target inoculum size. After adding the matrix, a brief vortex mixing was performed. At this point the magnetic beads were added, but mixed gently before addition.

[0200] The magnetic beads used were conjugated to the peps6 peptide, a synthetic version of the ApoH protein, a human protein present in blood and considered a potentially infectious product (MP10031, ApoH Technologies). The conditions for immunomagnetic separation using these beads were 30 min at 37 °C with gentle rotation (25 rpm) and a 20 degree tilt. Once binding occurred, 4 mL of 1X TTGB buffer was added, a dilution bank was performed with 100 μL of sample, and an agar plate was used to determine the bacterial count before magnetic retention. The time required for accurate magnetic separation was set at approximately 15 min. The supernatant was then removed and four washes were performed. For each wash step, 5 mL of wash buffer (Mueller-Hinton cation-adjusted broth) was added, vortexed for 1 min, the sample was incubated at 37 °C for 2 min with gentle rotation (25 rpm), the tube was briefly placed on a magnet (5 min) to recover the beads, and the supernatant was discarded by pipetting.

[0201] After washing, the beads were resuspended in 500 μL of culture medium (Mueller-Hinton Cation Adjusted Broth), vortexed for 1 min, and 250 μL of each sample was transferred to two 1.5 mL tubes. At this point, a second treatment with the reagent was performed to further wash the samples and disperse any aggregates that may have been detected in the previous assay. Specifically, 10 μL of protease was added and allowed to act for 30 seconds. To avoid the action of the protease on the bacteria, the protease was removed by centrifugation at 1000 g force for 1 min using a fixed angle rotor. Finally, the supernatant was discarded and the beads were resuspended in 500 μL of (Mueller-Hinton Cation Adjusted Broth) and vortexed for 1 min. A bank dilution was performed using 100 μL of matrix and all samples were plated on agar plates, and the samples were also observed under a microscope. Figure 27 shows images of the samples with and without protease.

[0202] 5 μL of each sample was transferred to a 384-well plate containing dye (SYBR-Green + PI), incubated at room temperature for 10 min, and centrifuged (3000 g force, 1 min) before visualization.

[0203] Table 2 below shows that significant numbers of bacteria were recovered at the end of the washing process, and that bacterial counts were higher when protease was used after washing, likely due to the dispersion of bacterial aggregates.

[0204] [Table 2]

[0205] Examples of computer systems: FIG. 20 is a block diagram of exemplary components of a computer system 2000. One or more computer systems 2000 may be used to implement, for example, any of the embodiments described herein, and combinations and subcombinations thereof. In some embodiments, one or more computer systems 2000 may be used for image acquisition, image analysis, and data processing, such as the microscope or processing device 116 in the AST subsystem 1700, as described herein. In some embodiments, one or more computer systems 2000 may be used in the controller 109 to program and operate the operation of various components in the analysis device 200. The computer system 2000 may include one or more processors (also referred to as central processing units, or CPUs), such as processor 2004. The processor 2004 may be connected to a communication infrastructure or bus 2006.

[0206] The computer system 2000 also includes a user input / output interface 2002 , such as a monitor, keyboard, pointing device, etc., and can communicate with a communications infrastructure 2006 via user input / output devices 2003 .

[0207] One or more of the processors 2004 may be a graphics processing unit (GPU). In one embodiment, a GPU may be a processor that is a specialized electronic circuit designed to handle mathematically intensive applications. GPUs may have a parallel structure that is efficient for parallel processing of large blocks of data, such as mathematically intensive data common in computer graphics applications, images, videos, etc.

[0208] The computer system 2000 may also include a main or primary memory 2008, such as a random access memory (RAM). The main memory 2008 may include one or more levels of cache. The main memory 2008 may store control logic (i.e., computer software) and / or data therein. In some embodiments, the main memory 2008 may include optical logic configured to perform sepsis detection, sepsis likelihood prediction, pathogen identification, and susceptibility testing, and may generate patient treatment recommendations accordingly.

[0209] The computer system 2000 may also include one or more secondary storage devices or memories 2010. The secondary memory 2010 may include, for example, a hard disk drive 2012 and / or a removable storage drive 2014.

[0210] The removable storage drive 2014 may interface with a removable storage unit 2018. The removable storage unit 2018 may include available or readable computer readable storage having stored thereon computer software (control logic) and / or data. The removable storage 2018 may be a program cartridge and cartridge interface (such as found in a video game device), a removable memory chip (such as an EPROM or PROM) and associated socket, a memory stick and USB port, a memory card and associated memory card slot, and / or other removable storage device and associated interface. The removable storage drive 2014 may read from and / or write to the removable storage 2018.

[0211] Secondary storage 2010 may include other means, devices, components, equipment or other approaches that allow computer programs and / or other instructions and / or data to be accessed by computer system 2000. Such means, devices, components, equipment or other approaches could include, for example, a removable storage unit 2022 and interface 2020. Examples of removable storage units 2022 and interfaces 2020 include program cartridges and cartridge interfaces (such as those found in video game devices), removable memory chips (such as EPROMs or PROMs) and associated sockets, memory sticks and USB ports, memory cards and associated memory card slots, and / or other removable storage devices and associated interfaces.

[0212] The computer system 2000 may further include a communication interface or network interface 2024. The communication interface 2024 may enable the computer system 2000 to communicate and interact with any combination of external devices, external networks, external entities, etc. (individually and collectively referred to by reference number 2028). For example, the communication interface 2024 may enable the computer system 2000 to communicate with external or remote devices 2028 via a communication path 2026, which may be wired and / or wireless (or a combination thereof) and include any combination of a LAN, a WAN, the Internet, etc. Control logic and / or data may be transmitted to or received from the computer system 2000 via the communication path 2026.

[0213] The computer system 2000 may be, but is not limited to, a personal digital assistant (PDA), a desktop workstation, a laptop or notebook computer, a netbook, a tablet, a smartphone, a smart watch, other wearable, a consumer electronics device, part of the Internet of Things (IOT), and / or an embedded system, or any combination thereof.

[0214] The computer system 2000 may be a client or a server and may be a remote or distributed cloud computing solution, a local or on-premise software (an "on-premise" cloud-based solution), an "as a service" model (e.g., Content as a Service (CaaS), Digital Content as a Service (DCaaS), Software as a Service (SaaS), Managed Software as a Service (MSaaS), Platform as a Service (PaaS), Desktop as a Service (DaaS), Framework as a Service (FaaS), Backend as a Service (BaaS), Mobile Backend as a Service (MBaaS), Infrastructure as a Service (IaaS), etc.), and / or a hybrid model including any combination of the foregoing examples or other service or delivery paradigms.

[0215] Applicable data structures, file formats, and schemas in the computer system 2000 may use JavaScript Object Notation (JSON), Extensible Markup Language (XML), Yet Another Markup Language (YAML), Extensible Hypertext Markup Language (XHTML), Wireless Markup Language (WML), MessagePack, XML User Interface Language (XUL), or other functionally similar representations, either alone or in combination, or may use proprietary data structures, formats, or schemas exclusively or in combination with known or open standards.

[0216] In some embodiments, tangible, non-transitory devices or articles of manufacture including usable or readable, tangible, non-transitory computer media having control logic (software) stored thereon may also be referred to herein as computer program products or program storage devices. This includes, but is not limited to, computer system 2000, main memory 2008, secondary memory 2010, and removable storage units 2018 and 2022, as well as tangible articles of manufacture embodying any combination of the foregoing. Such control logic, when executed by one or more data processing devices (such as computer system 2000), may cause such data processing devices to operate as described herein.

[0217] Based on the teachings contained herein, it will be apparent to one skilled in the relevant art how to make and use embodiments of the present disclosure using data processing devices, computer systems, and / or computer architectures other than those shown in Figure 20. In particular, embodiments may operate with software, hardware, and / or operating system implementations other than those described herein.

[0218] It is to be understood that the Detailed Description section, and not the Summary and Abstract sections, is used to interpret the claims. The Summary and Abstract sections present one or more exemplary embodiments of the present disclosure as intended by the inventors, but are not exhaustive, and thus are not intended to limit the present disclosure and the appended claims in any manner.

[0219] The embodiments of the present disclosure are described above with the aid of functional building blocks illustrating the implementation of certain functions and relationships thereof. The boundaries of these functional building blocks have been arbitrarily defined herein for the convenience of description. Other boundaries may be defined as long as the certain functions and relationships thereof are appropriately performed.

[0220] The foregoing description of the specific embodiments sufficiently reveals the general nature of the disclosure, so that those skilled in the art can easily modify and / or adapt the specific embodiments to various applications without undue experimentation and without departing from the general concept of the disclosure. Such adaptations and modifications are therefore intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the terms and expressions in this specification are for the purpose of explanation and not for the purpose of limitation. The terms and expressions in this specification should be interpreted by those skilled in the art in light of the teaching and guidance.

[0221] The breadth and scope of the present disclosure should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.

Claims

1. receiving, by an analytical device, a sample preparation cartridge and a sample container, the sample container containing a sample including the pathogen; placing a first needle from the sample preparation cartridge into a pipettor system within the analytical device; inserting a first needle into a sample container using a pipettor system; transferring the sample from the sample container through a first needle to a processing tube in the sample preparation cartridge; concentrating and enriching the sample for pathogens in a processing tube using an analytical device to obtain an enriched sample in the processing tube; dispensing a plurality of aliquots of the enriched sample into a plurality of reaction wells in an antimicrobial susceptibility testing (AST) cartridge in the analytical device, each aliquot corresponding to a respective reaction well, each reaction well containing a predetermined concentration of an antimicrobial; incubating the aliquots in the reaction wells of the AST cartridge for a predetermined period of time to allow a reaction to occur between the pathogen in each reaction well and the antimicrobial agent in each reaction well; acquiring an image of each reaction well in the AST cartridge using a microscope in the analyzer; analyzing the images by a processor coupled to the microscope in the analysis device to determine the susceptibility of the pathogen in each reaction well to the antimicrobial agent in each reaction well; A method comprising:

2. The sample container is a blood culture bottle, In the blood culture bottle, the sample is incubated long enough for the pathogen to grow but not reach a plateau of growth. The method of claim 1.

3. the sample container is a blood sample tube; The method of claim 1.

4. further comprising identifying the number of pathogens in the transferred sample using a fluorescent dye and a microscope of the analyzer to label and count the pathogens; Depending on the identification results, enriching or diluting the transferred sample to obtain a predetermined number of pathogens in the enriched sample; The method of claim 1.

5. concentrating the pathogens in the transferred sample using an analytical device; transferring the processing tube to a centrifuge in an analytical device; centrifuging the processing tube in a centrifuge to concentrate pathogens in the transferred sample; and removing liquid from the processing tube using a pipettor system to leave concentrated pathogens in the processing tube. The method of claim 1.

6. the sample is a blood sample; The method includes, prior to centrifugation of the processing tubes, adding one or more lysis reagents to the processing tube; mixing one or more lytic reagents with the blood sample in the processing tube to lyse blood cells in the blood sample; The method of claim 5.

7. the one or more lysis reagents comprise one or more saponin-based buffers; The method of claim 6.

8. the one or more lysis reagents include one or more detergents, surfactants, or proteases; The method of claim 6.

9. and washing the concentrated pathogens after a predetermined additional time by centrifuging the processing tube and removing the supernatant to obtain an enriched sample. The method of claim 5.

10. the processing tube further comprising magnetic beads configured to attach to the concentrated pathogens in the processing tube; The method is using a magnet station of the analyzer to retain the concentrated pathogens attached to the magnetic beads in the processing tube; removing excess liquid from within the processing tube to result in an enriched sample containing concentrated pathogens; further comprising: The method of claim 5.

11. Magnetic beads are coated with a non-specific ligand, The method of claim 10.

12. Magnetic beads are coated with specific ligands specific to a particular pathogen in the concentrated pathogens in the processing tube. The method of claim 10.

13. The method further comprises the step of adding a treatment with protease and / or DNAse after attaching the concentrated pathogens to the magnetic beads and before retaining the concentrated pathogens attached to the magnetic beads with a magnet. The method of claim 10.

14. adding one or more wash materials to the concentrated pathogens in the processing tube to remove blood components or debris from the concentrated pathogens, leaving an enriched sample in the processing tube; The method of claim 5.

15. The one or more cleaning materials include a combination of one or more buffers, detergents, surfactants, and proteases; 15. The method of claim 14.

16. adding an antimicrobial agent in liquid form to each reaction well of the AST cartridge prior to dispensing multiple aliquots of the enriched sample into the reaction wells; The method of claim 1.

17. the antimicrobial agent in each reaction well of the AST cartridge is in a dried or lyophilized form prior to dispensing multiple aliquots of the enriched sample into the reaction wells; The method of claim 1.

18. Dispensing a plurality of aliquots into a plurality of reaction wells comprises: using a jet dispensing method to perform non-contact dispensing of the aliquot by piercing the septum of each of the reaction wells with a second needle of the sample preparation cartridge and without the second needle contacting the bottom wall of each of the reaction wells; The method of claim 1.

19. the second needle is similar to the first needle; 20. The method of claim 18.

20. the volume of each aliquot dispensed by the second needle ranges from about 0.5 μL to about 10 μL; 20. The method of claim 18.

21. The number of pathogens in the sample is less than 200, The method of claim 1.

22. the number of pathogens in the enriched sample ranges from about 1,000 to about 100,000; The method of claim 1.

23. The number of pathogens in the enriched sample is approximately 10,000. The method of claim 1.

24. Each reaction well includes a bottom wall having an inner surface and an outer surface, a reaction between the pathogen in the aliquot in each reaction well and the antimicrobial agent occurs above the inner surface of the bottom wall of each reaction well in the AST cartridge, and an image of the AST cartridge is acquired at the outer surface of the bottom wall of each reaction well of the AST cartridge. The method of claim 1.

25. The predetermined period for incubating the aliquot is about 2 hours or less. The method of claim 1.

26. After incubating the aliquots in each reaction well, the AST cartridge is transferred to a centrifuge in the analyzer, and the pathogens in each aliquot are transferred to the bottom wall of each reaction well for acquiring an image of the AST cartridge. The method of claim 1.

27. After incubating the aliquots in each reaction well, the method further includes using a magnet of the analyzer to move pathogens in each aliquot to the bottom wall of each reaction well to acquire an image of the AST cartridge, where the pathogens adhere to the magnetic beads in each aliquot; The method of claim 1.

28. adding a first fluorescent dye to each reaction well to stain the pathogens in the aliquot of the reaction well prior to acquiring the image; The method of claim 1.

29. adding a first fluorescent dye to each reaction well; using a jet dispensing method to perform non-contact dispensing of the first fluorescent dye by piercing the septum of each of the reaction wells with a second needle of the sample preparation cartridge and without the second needle contacting the bottom wall of each of the reaction wells; 29. The method of claim 28.

30. the volume of the first fluorescent dye dispensed by the second needle into each reaction well ranges from about 0.5 μL to about 10 μL; 30. The method of claim 29.

31. the second needle is similar to the first needle; 31. The method of claim 30.

32. acquiring the images includes acquiring one or more fluorescent images to detect the first fluorescent dye in each reaction well of the AST cartridge; 29. The method of claim 28.

33. Analyzing the image includes calculating the number of fluorescent pathogens in each reaction well in the AST cartridge.

33. The method of claim 32.

34. further comprising the step of adding a second fluorescent dye to each reaction well prior to acquiring the image.

29. The method of claim 28.

35. Within the aliquot of reaction wells, a first fluorescent dye comprises a DNA-binding dye that labels live cells within the aliquot of reaction wells, and a second fluorescent dye comprises a fluorescent intercalator that labels dead cells or does not permeate intact cell membranes; 35. The method of claim 34.

36. Determining pathogen susceptibility involves: determining whether one or more of the pathogens are susceptible, intermediate, or resistant to the antimicrobial agent in each reaction well based on staining of the cells in the aliquots with the first fluorescent dye and / or the second fluorescent dye; 36. The method of claim 35.

37. Determining that a particular pathogen is resistant to a particular antimicrobial agent in a reaction well can be accomplished by: determining, based on staining of cells in the aliquot with the first fluorescent dye and / or the second fluorescent dye, that the ratio of the number of dead cells to the number of live cells is below a predetermined threshold for a particular antimicrobial agent at a predetermined concentration; 37. The method of claim 36.

38. Determining pathogen susceptibility involves: determining whether one or more of the pathogens are susceptible, intermediate, or resistant to the antimicrobial agent in each reaction well based on staining of the cells in the aliquot with a first fluorescent dye; The method of claim 1.

39. a first reaction well of the plurality of reaction wells containing a first concentration of an antimicrobial agent; The first concentration is a high dose that is significantly higher than the clinical breakpoint for the antimicrobial agent and the pathogen; The method of claim 1.

40. Determining pathogen susceptibility involves: comparing the image of the first reaction well with a control to determine highly resistant pathogens within about one hour; 40. The method of claim 39.

41. determining, by the processor, a minimum inhibitory concentration (MIC) of a particular antimicrobial agent for inhibiting growth of a particular pathogen among the one or more pathogens based on the image of the AST cartridge. The method of claim 1.

42. determining, by the processor, whether the particular pathogen is susceptible, intermediate, or resistant to the particular antimicrobial agent by analyzing a set of rules in a database communicatively coupled to the processor; 42. The method of claim 41.

43. 1. An antimicrobial susceptibility testing (AST) cartridge comprising: a base including a plurality of reaction wells, each reaction well including a bottom wall, the bottom wall being optically transparent; a septum disposed on the base to seal each reaction well in the plurality of reaction wells; a cover disposed on the septum; each reaction well in the plurality of reaction wells containing a predetermined concentration of an antimicrobial agent for reacting with a respective aliquot of the enriched sample containing the pathogen; The antimicrobial agent is disposed within each reaction well. AST cartridge.

44. The antimicrobial agent in each reaction well is in liquid form.

44. The AST cartridge of claim 43.

45. The antimicrobial agent in each reaction well is in a dry or lyophilized form.

44. The AST cartridge of claim 43.

46. a bottom wall of each reaction well of the plurality of reaction wells configured for optical interrogation; 44. The AST cartridge of claim 43.

47. a bottom wall of each reaction well of the plurality of reaction wells configured for a fluorescent microscope; 44. The AST cartridge of claim 43.

48. The diameter of the bottom wall of each reaction well is less than about 2 millimeters; 44. The AST cartridge of claim 43.

49. Each reaction well comprises a cone; 44. The AST cartridge of claim 43.

50. each reaction well is configured to receive an aliquot of the enriched sample containing the pathogen by non-contact dispensing with a needle by piercing a septum of the reaction well without the needle contacting a bottom surface of each reaction well; 44. The AST cartridge of claim 43.

51. the cover includes a plurality of openings; each opening aligned with a respective reaction well of a plurality of reaction wells in the base; 44. The AST cartridge of claim 43.

52. the plurality of reaction wells are configured to fit into corresponding wells in the temperature control block; a temperature control block for heating the plurality of reaction wells; 44. The AST cartridge of claim 43.

53. the base, septum, and cover each include an opening in the center of the AST cartridge; The opening is adapted for insertion by a pipette tip to transfer the AST cartridge.

44. The AST cartridge of claim 43.

54. the diaphragm is overmolded onto the cover to form a combined part; The combined parts are assembled onto the base by at least one of a snap fit connection or a mechanical fastener; 44. The AST cartridge of claim 43.

55. The septum is a unitary structure that extends across multiple reaction wells; 44. The AST cartridge of claim 43.

56. The diaphragm includes a plurality of assembled parts; Each part covers a reaction well.

44. The AST cartridge of claim 43.

57. the diaphragm comprises at least one of rubber, polytetrafluoroethylene (PTFE), thermoplastic elastomer (TPE), silicone, butyl rubber, or a combination thereof; 44. The AST cartridge of claim 43.

58. The diaphragm is composed of a double layer of polytetrafluoroethylene (PTFE) and another material selected from the group consisting of silicone, rubber, and butyl rubber.

44. The AST cartridge of claim 43.

59. The diaphragm has a thickness in the range of about 1 to 2 mm.

44. The AST cartridge of claim 43.

60. the plurality of reaction wells comprises about 100 reaction wells; Each reaction well holds a volume of approximately 30 μL.

44. The AST cartridge of claim 43.

61. The cover is made of polypropylene (PP) or polycarbonate (PC) material.

44. The AST cartridge of claim 43.

62. The base is made of polystyrene (PS), 44. The AST cartridge of claim 43.

63. the cover includes an identifier that is scanned by an analyzer to perform antimicrobial susceptibility testing; 44. The AST cartridge of claim 43.

64. the identifier is a data matrix or a barcode, 64. The AST cartridge of claim 63.

65. 1. A system for enriching a sample, comprising: a housing configured to receive a sample container containing a sample including a pathogen; a pipettor system disposed within the housing; one or more centrifuges disposed within the housing; a controller; The controller transferring at least a portion of the sample from the sample container to a processing tube using a pipettor system; centrifuging the processing tubes using one or more centrifuges to concentrate pathogens in the samples; Remove fluid from the processing tube using a pipettor system, leaving concentrated pathogens in the processing tube; adding growth media to the concentrated pathogens in the processing tube using a pipettor system; and allowing the concentrated pathogens in the processing tube to grow for a predetermined period of time; and washing the concentrated pathogens after a predetermined period of time to obtain an enriched sample in the processing tube. system.

66. The sample container is a blood culture bottle, and the sample in the blood culture bottle is incubated for a period of time that allows pathogen growth but does not reach a plateau of growth.

66. The system of claim 65.

67. The sample container is a blood sample tube.

66. The system of claim 65.

68. further comprising a mixer disposed within the housing; the sample is a blood sample, The controller, prior to centrifugation of the processing tube, adding one or more lysis reagents to the processing tube using a pipettor system; configured to mix one or more lysis reagents with the blood sample in the processing tube using the mixer to lyse blood cells in the blood sample; 66. The system of claim 65.

69. the one or more lysis reagents include one or more saponin-based buffers, detergents, surfactants, or proteases; 69. The system of claim 68.

70. Washing the enriched pathogens includes centrifuging the processing tube using one or more centrifuges and removing the supernatant from the processing tube using a pipettor system to leave the enriched sample in the sample tube.

66. The system of claim 65.

71. further comprising an antimicrobial susceptibility testing (AST) subsystem; the processing tube further comprises magnetic beads configured to attach to the concentrated pathogens in the processing tube; the AST subsystem is configured to apply a magnetic force to the processing tube to retain the concentrated pathogens attached to the magnetic beads in the processing tube; The controller is further configured to remove excess water from the processing tube using the pipettor system, resulting in an enriched sample containing concentrated pathogens.

66. The system of claim 65.

72. Magnetic beads are coated with a non-specific ligand, 72. The system of claim 71.

73. Magnetic beads are coated with specific ligands specific to a particular pathogen in the concentrated pathogens in the processing tube.

72. The system of claim 71.

74. the controller is further configured to apply one or more wash materials to the concentrated pathogens in the processing tube to wash and remove blood components or debris from the concentrated pathogens, leaving an enriched sample in the processing tube; 66. The system of claim 65.

75. The one or more cleaning materials include a combination of one or more buffers, detergents, surfactants, and proteases; 75. The system of claim 74.

76. the sample container and the processing tube each include a septum that allows insertion of a first needle coupled to a first pipettor of the pipettor system; 66. The system of claim 65.

77. The step of transferring at least a portion of the sample from the sample container to the processing tube includes inserting a first needle through a septum of the processing tube and dispensing at least a portion of the sample through the first needle into the processing tube; 77. The system of claim 76.

78. a receptacle configured to receive an antimicrobial susceptibility testing (AST) cartridge containing a plurality of reaction wells; a microscope configured to acquire one or more images of the concentrated pathogens in the enriched sample after transferring the enriched sample to the plurality of reaction wells; further comprising:

66. The system of claim 65.

79. One or more centrifuges a first centrifuge configured to hold and centrifuge the processing tubes; a second centrifuge configured to hold and centrifuge the AST cartridge in a vertical orientation; 79. The system of claim 78.

80. a magnet station configured to apply a magnetic force to move the concentrated pathogens to a bottom surface of a reaction well in the AST cartridge; The concentrated pathogens are attached to magnetic beads.

79. The system of claim 78.

81. 1. A system for analyzing a sample, comprising: The system is a housing configured to receive a processing tube and an antimicrobial susceptibility testing (AST) cartridge; the AST cartridge includes a plurality of reaction wells, each reaction well including a bottom wall, the bottom wall being optically transparent; The system further a heater disposed within the housing; a pipettor system disposed within the housing; a microscope disposed within the housing; a controller; The controller configured to dispense a plurality of aliquots of the enriched sample containing the pathogen from the processing tube into a plurality of reaction wells in the AST cartridge using a pipettor system; Each aliquot corresponds to a different reaction well. Each reaction well contains a predetermined concentration of antimicrobial agent; The controller further using a heater to incubate the aliquots in the reaction wells of the AST cartridge for a predetermined period of time to allow a reaction to occur between the pathogens and the antimicrobial agent in each reaction well; using a microscope to obtain one or more images of the bottom wall of each reaction well in the AST cartridge; and a processor coupled to the microscope configured to analyze the one or more images to determine the susceptibility of the pathogen in each reaction well to the antimicrobial agent. system.

82. the controller is further configured to add, using the pipettor system, a first fluorescent dye to each reaction well to stain the pathogens in the aliquot of the reaction well before acquiring the one or more images; 82. The system of claim 81.

83. acquiring one or more images includes acquiring one or more fluorescent images to detect the first fluorescent dye in each reaction well of the AST cartridge; 83. The system of claim 82.

84. Analyzing the one or more images includes calculating the number of fluorescent pathogens in each reaction well in the AST cartridge.

84. The system of claim 83.

85. the controller is further configured to add, using the pipettor system, a second fluorescent dye to each reaction well before acquiring the one or more images.

83. The system of claim 82.

86. Within the aliquot of reaction wells, a first fluorescent dye comprises a DNA binding dye that labels live cells within the aliquot of reaction wells, and a second fluorescent dye comprises a fluorescent intercalator that labels dead cells; 86. The system of claim 85.

87. 1. A method for manufacturing an antimicrobial susceptibility testing (AST) cartridge, comprising: manufacturing a cover with a plurality of openings; overmolding a diaphragm onto the cover, the first side of the diaphragm extending across the plurality of openings; fabricating a base comprising a plurality of reaction wells; attaching a base to a second side of the septum, the second side of the septum extending across and sealing the plurality of reaction wells of the base; method.

88. The cover includes polypropylene (PP) or polycarbonate (PC).

88. The method of claim 87.

89. The base comprises polystyrene (PS), 88. The method of claim 87.

90. adding a predetermined concentration of antimicrobial agent in liquid form to each reaction well before attaching the base to the second side of the septum; drying the antimicrobial agent in each reaction well to a dry or lyophilized state using forced air; further comprising:

88. The method of claim 87.

91. The steps of adding and drying the antimicrobial agent in each reaction well are completed within a predetermined time to prevent degradation of the antimicrobial agent; 91. The method of claim 90.

92. The predetermined time is about 15 minutes.

92. The method of claim 91.

93. The predetermined time is about 10 minutes.

92. The method of claim 91.

94. Manufacturing the cover involves the use of injection molding, 88. The method of claim 87.

95. and attaching the base to the second side of the diaphragm includes using at least one of a snap fit connection or a mechanical fastener.

88. The method of claim 87.

96. further comprising the step of aligning each opening in the cover with a respective reaction well of the plurality of reaction wells in the base when attaching the base to the second side of the septum; 88. The method of claim 87.

97. The step of fabricating the base includes fabricating a plurality of reaction wells connected to one another as a single component; 88. The method of claim 87.

98. the diaphragm comprises at least one of rubber, polytetrafluoroethylene (PTFE), thermoplastic elastomer (TPE), silicone, butyl rubber, or a combination thereof; 88. The method of claim 87.

99. The diaphragm is composed of a double layer of polytetrafluoroethylene (PTFE) and another material selected from the group consisting of silicone, rubber, and butyl rubber.

88. The method of claim 87.

100. The step of installing the first needle in the pipette system includes attaching a proximal end of a plastic body of the first needle to the pipette system; The method of claim 1.

101. The plastic body includes an aerosol filter configured to prevent contamination within the pipettor system. The method of claim 100.

102. The plastic body includes a predetermined number of slots configured to provide ventilation between the interior and exterior of the sample tube. The method of claim 100.

103. The first needle includes a stainless steel cannula attached to a plastic body. The method of claim 100.

104. The first needle further includes a secondary cannula disposed about the inner core of the first needle; the secondary cannula includes one or more vent holes; The method of claim 103.

105. The step of inserting the first needle into the sample tube includes inserting the first needle through a septum of the sample tube; The septum provides an airtight seal for the sample tube. The method of claim 100.

106. The step of transferring the sample includes installing the first needle in the pipetter system by attaching the proximal end of the plastic body of the first needle to the pipetter system; 66. The system of claim 65.

107. The step of transferring the sample further includes inserting the first needle into the processing tube by inserting the first needle through a septum of the processing tube; The septum provides an airtight seal to the process tube.

107. The system of claim 106.

108. The plastic body includes an aerosol filter configured to prevent contamination within the pipettor system.

108. The system of claim 107.

109. The plastic body includes a predetermined number of slots configured to provide ventilation between the interior and exterior of the sample tube.

108. The system of claim 107.

110. The first needle includes a stainless steel cannula attached to a plastic body.

108. The system of claim 107.

111. The first needle further includes a secondary cannula disposed about the inner core of the first needle; the secondary cannula includes one or more vent holes; 111. The system of claim 110.

112. The step of dispensing the multiple aliquots includes installing the first needle in the pipetter system by attaching the proximal end of the plastic body of the first needle to the pipetter system; 82. The system of claim 81.

113. The step of dispensing the plurality of aliquots further comprises inserting a first needle into the processing tube by inserting the first needle through a septum of the processing tube; The septum provides an airtight seal to the process tube.

113. The system of claim 112.

114. The plastic body includes an aerosol filter configured to prevent contamination within the pipettor system.

114. The system of claim 113.

115. The plastic body includes a predetermined number of slots configured to provide ventilation between the interior and exterior of the sample tube.

114. The system of claim 113.

116. The first needle includes a stainless steel cannula attached to a plastic body.

114. The system of claim 113.

117. The first needle further includes a secondary cannula disposed about the inner core of the first needle; the secondary cannula includes one or more vent holes; 117. The system of claim 116.

Images