Systems, methods, and devices for antimicrobial sensitivity testing

Abstract

Described herein are systems, methods, and devices for antimicrobial sensitivity testing (AST) directly from a blood sample. A system for enriching a sample includes a housing configured to receive a sample container, a sample preparation cartridge, and an AST cartridge for processing and testing a sample using one or more centrifuges, a pipetting system, a controller, an AST subsystem, a magnet station, a microscope, and a heater disposed inside the housing. The system is configured for transferring a sample from a blood sample container into a processing tube in a sample preparation cartridge using a septum and needle based pipetting system. After the sample is transferred, the system performs the steps of separating, enriching and concentrating pathogens in the sample for rapid detection. The system then dispenses an aliquot of the enriched sample into reaction wells containing the antimicrobial agent in the AST cartridge, acquires an image of each reaction well after incubation, and determines the sensitivity of the pathogen to the antimicrobial agent based on analyzing the acquired images.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 299,555, filed January 14, 2022, which is incorporated herein by reference in its entirety. This application is related to co-pending U.S. Patent Application No. _____________, filed January 13, 2023, entitled “Systems, Methods, and Devices for Pathogen Identification” (Attorney Docket No. 4518.003PC01) 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 entirety. Background Art Technical Field

[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 treatment for a patient. Background Art

[0003] Sepsis is defined as a life-threatening organ dysfunction caused by a dysregulated host response to infection. Patients who experience or suffer from sepsis may begin with a local infection, such as pneumonia, that causes inflammation of the body caused by the patient's immune system going into overdrive. If untreated, the inflammation may ultimately lead to organ failure and death of the patient. Sepsis causes 11 million deaths each year, many of which could be prevented through early diagnosis, appropriate clinical management, and treatment.

[0004] Sepsis diagnosis can be made by running laboratory tests to culture a blood sample for identification of infection. Current testing times can take several days to get results from the laboratory because time is required to obtain and process blood cultures of the bacteria that cause sepsis to ultimately determine their sensitivity to effective antimicrobial agents for treatment. However, sepsis testing can be time-sensitive for patients in the hospital because sepsis can lead to septic shock and death within hours if not properly identified and treated promptly. In addition, blood culture tests are slow and may not consistently provide reliable bacterial or fungal test results in patients who are clinically suspected of having sepsis, especially for patients who have been subjected to antibiotic therapy. The yield of obtaining positive blood cultures for patients is generally low, and even if a positive blood culture is not identified, the patient may be experiencing sepsis.

[0005] Traditional and automated methods for performing antimicrobial susceptibility testing require "pure culture" bacterial isolates and long periods of time (e.g., 6-24 hours) incubation for organism growth. Such conventional systems may be limited because they are growth-based, slow, expensive, require manual manipulation, and are not integrated. Without improved solutions, patients may continue to suffer and often get worse as doctors treat with empirical antibiotics while waiting for more actionable information from the laboratory about one or more causative agents (e.g., pathogens) of infection and which antimicrobial agents to use to treat the highly resistant pathogens of patients who are experiencing 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 in order to provide patients with appropriate treatment approaches for better patient outcomes.

[0007] This paper describes the system, method and device for directly carrying out antimicrobial sensitivity test (AST) from blood sample or other samples (such as urine, sterile body fluids, etc.). In the embodiment presented herein, AST system, analyzer device, AST box, sample preparation box and treatment tube are provided, to carry out fast AST from sample when there is no blood culture, while retaining the sample quality of sample. In some embodiments, single cell microscopy can be utilized to identify the phenotypic sensitivity of pathogen, thereby producing the diagnostic approach for fast and effective antimicrobial treatment. In some embodiments, the system, method and device for AST described herein can be used for treating patients with sepsis and / or other potential diseases.

[0008] In the embodiments, an exemplary method for performing a sensitivity test is described. The method includes: receiving a sample preparation box and a sample container by an analyzer device, the sample container containing a sample containing a pathogen; installing a first needle from the sample preparation box in a pipette system in the analyzer device; inserting the first needle into the sample container using the pipette system; transferring the sample from the sample container to a processing tube in the sample preparation box through the first needle; concentrating and enriching the pathogens in the sample in the processing tube using the analyzer device, thereby producing an enriched sample in the processing tube; and distributing a plurality of aliquots of the enriched sample to a plurality of reaction wells in an antimicrobial susceptibility test (AST) box in the analyzer device, wherein each aliquot corresponds to a corresponding reaction well, and wherein each reaction well contains a predetermined concentration of an antimicrobial agent. The method further includes incubating the aliquots in the reaction wells of the AST box for a predetermined period of time to allow a reaction between the pathogens in the aliquots and the antimicrobial agent in each reaction well; acquiring an image of each reaction well in the AST box by using a microscope in the analyzer device; and determining the sensitivity of the pathogen to the antimicrobial agent in each reaction well by analyzing the image through a processor coupled to the microscope in the analyzer device.

[0009] In another embodiment, an exemplary antimicrobial susceptibility test (AST) box is described. The AST box includes a base comprising a plurality of reaction wells, each reaction well comprising a bottom wall, wherein the bottom wall is optically transparent. The AST box further includes a diaphragm arranged on the base, the diaphragm seals each reaction well in the plurality of reaction wells, and a cover member arranged on the diaphragm. Each reaction well in the plurality of reaction wells accommodates an antimicrobial of a predetermined concentration for reacting with a corresponding aliquot of an enriched sample comprising a pathogen, and the antimicrobial is arranged in each reaction well.

[0010] In another embodiment, an exemplary system for enriching a sample is described. The system includes: a housing configured to receive a sample container containing a sample containing a pathogen; a pipette system disposed inside the housing; one or more centrifuges disposed inside the housing; and a controller. The controller is configured to transfer the sample from the sample container to a processing tube using the pipette system; centrifuge the processing tube using the one or more centrifuges to concentrate the pathogens in the sample; remove fluid from the processing tube using the pipette system, leaving concentrated pathogens in the processing tube; and add growth medium to the concentrated pathogens in the processing tube using the pipette system to grow the concentrated pathogens in the processing tube for a predetermined period of time; and clean the concentrated pathogens after the predetermined period of time to obtain an enriched sample in the processing tube.

[0011] In another embodiment, an exemplary system for analyzing a sample is described. The system includes a housing configured to receive a processing tube and an antimicrobial susceptibility test (AST) box. The AST box includes a plurality of reaction wells, each reaction well comprising a bottom wall, wherein the bottom wall is optically transparent. The system further includes a heater, the heater is arranged inside the housing; a pipette system, the pipette system is arranged inside the housing; a microscope, the microscope is arranged inside the housing; and a controller. The controller is configured to use the pipette system to distribute a plurality of aliquots of an enriched sample containing a pathogen from the processing tube to a plurality of reaction wells in the AST box, wherein each aliquot corresponds to a corresponding reaction well, and each reaction well contains a predetermined concentration of an antimicrobial agent. The controller is further configured to incubate aliquots in the reaction wells of the AST box for a predetermined period of time using the heater to allow a reaction to occur between the pathogen and the antimicrobial agent in each reaction well; acquire one or more images of the bottom wall of each reaction well in the AST box using the microscope; and determine, by a processor coupled to the microscope, the sensitivity of the pathogen to the antimicrobial agent in each corresponding reaction well by analyzing the one or more images.

[0012] In another embodiment, an exemplary method for manufacturing an AST box is described. The method includes: manufacturing a cover comprising a plurality of openings; overmolding a diaphragm into the cover, wherein a first side of the diaphragm extends across the plurality of openings; creating a base comprising a plurality of reaction holes; and attaching the base to a second side of the diaphragm, wherein the second side of the diaphragm extends across and seals the plurality of reaction holes in the base.

[0013] The further features and advantages and 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 restrictive. Such embodiments are presented herein for illustrative purposes only. Based on the teachings contained herein, additional embodiments will be clear to one or more technicians in related fields. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0015] Figure 1 A diagram of a system for performing antimicrobial susceptibility testing (AST) according to an embodiment of the present disclosure is shown.

[0016] Figure 2 A diagram of an analyzer apparatus according to an embodiment of the present disclosure is shown.

[0017] Figure 3A A diagram showing a front view of an analyzer device according to an embodiment of the present disclosure.

[0018] Figure 3B A diagram showing a top view of an analyzer device according to an embodiment of the present disclosure is shown.

[0019] Figure 4 A diagram of an analyzer device having three open compartments according to an embodiment of the present disclosure is shown.

[0020] Figure 5 A diagram of a sample preparation kit according to an embodiment of the present disclosure is shown.

[0021] Fig. 6A A diagram showing processing tubes and other components within a sample preparation cartridge according to an embodiment of the present disclosure is shown.

[0022] Figure 6B A diagram showing processing tubes and other components within a linear sample preparation cartridge according to an embodiment of the present disclosure is shown.

[0023] Fig. 7A and Figure 7B A diagram of a processing tube according to an embodiment of the present disclosure is shown.

[0024] Fig. 8A , Figure 8B and Figure 8C A diagram of a needle configured for insertion into a treatment tube is shown in accordance with an embodiment of the present disclosure.

[0025] Fig. 9A and Fig. 9B Diagrams of a high volume needle and a low volume needle are shown, respectively, according to embodiments of the present disclosure.

[0026] Fig.10 A diagram showing an example of a high capacity needle according to an embodiment of the present disclosure is shown.

[0027] Fig.11 A diagram of a low volume needle docked with a sample preparation cartridge is shown according to an embodiment of the present disclosure.

[0028] Fig. 12A and Fig. 12B A diagram of an AST cassette according to an embodiment of the present disclosure is presented.

[0029] Fig.13 A diagram showing a low volume needle inserted into an AST cassette according to an embodiment of the present disclosure is shown.

[0030] Fig.14A and Fig. 14B A diagram showing an exemplary centrifuge for use in an analyzer according to an embodiment of the present disclosure is shown.

[0031] Fig.15A , Fig. 15B , Fig. 15C and Fig.15D A diagram showing an exemplary enrichment subsystem for use in an analyzer according to an embodiment of the present disclosure is shown.

[0032] Fig.16A , Fig. 16B and Fig. 16C A diagram showing an exemplary mechanical device for use in an analyzer according to an embodiment of the present disclosure is shown.

[0033] Fig.17A and Fig. 17B A diagram of an AST cassette interfaced with an imaging subsystem in an analyzer according to an embodiment of the present disclosure is shown.

[0034] Fig.18 A flow chart of a method for performing AST of a sample according to an embodiment of the present disclosure is shown.

[0035] Fig.19 A flow chart of a method for making or assembling an AST cassette according to an embodiment of the present disclosure is shown.

[0036] Fig. 20 A block diagram is shown of exemplary components of a computer system according to an embodiment of the present disclosure.

[0037] Figure 21- Fig. 27Experimental results from testing based on embodiments of the present disclosure are presented.

[0038] Embodiments of the present disclosure will be described with reference to the accompanying drawings. DETAILED DESCRIPTION

[0039] Although specific configurations and arrangements are discussed, it should be understood that this is done for illustrative purposes only. Those skilled in the relevant art will recognize that other configurations and arrangements may be used without departing from the spirit and scope of the present disclosure. It will be clear to those skilled in the relevant art that the present disclosure may also be used in various other applications.

[0040] Units, prefixes and symbols are all expressed in the form approved by the International System of Units (SI). Numerical ranges include numbers that define the ranges. In the case of enumerated value ranges, it is understood that each intermediate integer value and fraction thereof between the upper and lower limits of the enumerated ranges, and each subrange between these values ​​are also specifically disclosed. Therefore, the ranges enumerated herein are understood to be shorthand for all values ​​(including the enumerated endpoints) within the range. For example, a range of 1 to 10 is understood to include: any number, combination of numbers, or subranges from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0041] Where values ​​are explicitly enumerated, it is understood that values ​​of approximately the same quantity or amount as the enumerated value are also within the scope of the present disclosure. Where combinations are disclosed, each subcombination of the elements of the combination is also specifically disclosed and within the scope of the present disclosure. Conversely, where different elements or groups of elements are disclosed individually, their combinations are also disclosed.

[0042] The use of alternatives (e.g., "or") should be understood to mean one, both, or any combination of the alternatives. As used herein, the indefinite article "a" or "an" should be understood to mean "one or more" of any stated or listed components.

[0043] The term "about" refers to a value or composition within an acceptable error range for a particular value or composition determined by one of ordinary skill in the art, which depends in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, according to the practice in the art, "about" can mean within 1 or more than 1 standard deviation. Alternatively, "about" can mean a range of up to 10%. In addition, particularly with respect to biological systems or processes, the term can mean up to an order of magnitude or up to 5 times of a value. When specific values ​​or compositions are provided in the present application and claims, unless otherwise stated, it should be assumed that the meaning of "about" is within an acceptable error range for the specific value or composition.

[0044] As described herein, unless otherwise indicated, any concentration range, percentage range, ratio range or integer range should be understood to include any integer and, where appropriate, fractional values ​​thereof (such as tenths and hundredths of an integer) within the recited range. introduce:

[0045] The current standard of care for detecting and treating sepsis relies on blood culture, which has an average detection time of about 13 hours. The blood culture test provides the organism without the identification (ID) of the pathogen, and then the positive is plated on the culture dish. In the conventional blood culture process, each adult patient takes two blood culture sets, each of which consists of an aerobic bottle and an anaerobic bottle to ensure that the entire spectrum of sepsis pathogenic bacteria is captured during the culture event. Typically, each culture is obtained from a separate venipuncture (e.g., the left and right arms of the patient). This is to ensure that bacterial shedding events are captured by culture so that bacteria can be "recovered" for downstream testing (e.g., ID and AST). After cultivation, aerobic bottles and anaerobic bottles are incubated in a blood culture instrument, where their growth is monitored in real time. Aerobic bottles and anaerobic bottles are incubated and stirred until any bacteria are allowed to undergo a hysteresis-logarithmic growth transition detected by electronics. Then, laboratory staff may be warned that the patient has a positive culture. Typically, blood cultures for most bacteria will become positive within an average of about 13 hours, while some yeasts and fungi may take longer (e.g., up to 5 days). However, many cultures are negative due to collection errors, insufficient blood volume obtained during collection, delayed transport to the laboratory, insufficient sensitivity, etc.

[0046] Due to the urgent nature of sepsis, after positive, the laboratory can immediately start inspection work to identify Gram bacteria staining (for example, Gram-positive or Gram-negative), determine important organisms, pollutants, single microbial infections or polymicrobial infections, and report intermediate information to the caregiver. In addition, the laboratory can take steps immediately to identify bacteria using rapid methods such as molecular diagnostic systems (which may require 1.5 hours to provide results). These systems can provide limited molecular information about the genetic drug resistance information of certain bacteria that exhibit these spectra. Alternatively, the laboratory can process positive blood culture (PBC) aliquots with matrix-assisted laser desorption / ionization time of flight (MALDI-TOF) mass spectrometry systems in about one hour to report ID. Using ID, the caregiver can confirm or potentially adjust antibiotics that may have been prophylactically applied to the patient. However, by the time of identifying bacteria, up to 20-24 hours (at most) may have passed since the patient first cultured.

[0047] While obtaining and reporting the identified pathogens, antimicrobial susceptibility testing (AST) can also be performed to determine the antibiotic sensitivity spectrum of the pathogen. In some cases, AST can be performed in vitro on isolated bacterial pathogens using manual methods, including microbroth dilution methods or culture-based assays (e.g., disk diffusion antibiotic susceptibility testing or using culture dish culture media for plating). In other cases, AST can be performed using an automated device with an antimicrobial resistance kit to test the minimum inhibitory concentration (MIC) or resistance of antimicrobials.

[0048] However, these systems may need isolated bacterial colonies. For example, the current method for AST may need a significant amount of clean bacteria to properly operate. The subculture from positive blood culture must first grow on the culture medium culture dish of the plate. For laboratories that run around the clock, this process may require 6-12 hours, but for laboratories that close overnight, it may sometimes take up to 24 hours. Then before being inoculated into the AST system, it may be necessary to adjust the uniformity of the culture (for example, taking 10,000 cells for input). After loading, AST analysis may require between 8-16 hours to report the antimicrobial sensitivity or drug resistance information of all organism categories. In addition, AST may not be started until at least the second day of the patient's hospitalization. In other words, the result analyzed from AST (for example, including whether bacteria are sensitive, moderately sensitive or resistant (SIR), MIC, or drug resistance information to specific antibiotics) can be reported to the caregiver about 2.5-3 days after the initial collection of the original blood culture sample from the patient.

[0049] The whole process may take several days to report key antibiotic information for sepsis patients. For example, if an infection is suspected, samples of blood, urine, sputum, etc. are collected from the patient and provided to the clinical laboratory to first determine whether there is an infectious agent. This may require 18-24 hours (e.g., day 1) to fully grow most pathogenic bacterial species. If the bacteria are isolated, another 18-24 hours (e.g., day 2) are further required to culture the isolate, and another 2-48 hours (e.g., day 3+) are required to identify the bacterial isolate and perform AST. Traditional and automated AST methods require "pure culture" bacterial isolates, and long periods of time (e.g., 6-24 hours) of incubation for organism growth. Conventional systems may be limited because they are based on growth, slow, expensive, require manual manipulation, and are not integrated. In the absence of an improved solution, while waiting for more feasible information about one or more causative factors (e.g., pathogens) of infection from the laboratory, the patient may continue to suffer and often get worse because the doctor treats with empirical antibiotics.

[0050] Current technology does not provide an integrated and comprehensive solution for the entire workflow of host response detection, pathogen identification, and antimicrobial or antibiotic susceptibility testing (AST). In some cases, some systems focus only on identifying a single aspect of the sepsis cascade, such as detection of the host response or detection of the pathogen. For example, a system can observe the host response for early indications of sepsis by detecting molecular leukocyte RNA markers (detecting gene expression via reverse transcription (RT)-PCR), but will not provide answers for pathogen identification or sensitivity. Immune response results can alert the caregiver that the patient is entering or has entered the sepsis cascade and urgently needs treatment or intervention to prevent further possibilities of irreversible morbidity. The caregiver can respond immediately by finding the site of infection and the infectious agent using traditional methods (such as by obtaining blood cultures from the site of infection to identify the infectious agent).

[0051] In terms of pathogen detection, current technology can provide detection and identification methods directly from blood, using PCR performed from blood samples, and then detect. However, such systems may be expensive, have limited menu options, and are difficult to serve when rapid AST results are not provided but limited molecular genetic resistance set solutions are provided. Other systems can utilize the rRNA RT-PCR of the pathogen directly from blood to carry out pathogen identification, but may also be limited to a limited menu (e.g., 15 targets or less). Ultimately, current technology does not provide automated rapid AST solutions directly from blood, but relies on positive blood cultures to test, which may require 13-20 hours to report any feasible matters. For example, some systems can obtain aliquots from positive blood culture (PBC) bottles, thereby saving the time (e.g., 6-24 hours) required for growing bacterial isolates from PBC bottles. However, these systems are limited to the quantity of drugs and organisms that they can report, and therefore have limited utility to health care providers. To significantly reduce morbidity and mortality, new diagnostic methods, devices, and systems are needed for rapidly determining antibiotic susceptibility and antibiotic resistance of bacteria causing infectious sepsis at the single cell or low copy number level, directly from blood samples without the significant time delay of multiple culture steps (e.g., bioamplification) currently required by standard of care methods.

[0052] Because sepsis conditions can often go undetected in patients and rapidly progress to life-threatening conditions, there is a clear need and demand for new and comprehensive systems, devices, and methods for rapidly testing patient samples and providing guidance on appropriate antimicrobial agents in order to save lives and reduce antimicrobial resistance (AMR). The systems, devices, and methods described herein provide a holistic and systematic approach for determining antibiotic susceptibility and resistance of pathogens in order to recommend effective and appropriate treatment for patients. AST System Overview:

[0053] Figure 1 A diagram of a system 101 for performing an antimicrobial susceptibility test (AST) according to an embodiment of the present disclosure is shown. 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 box 114, an AST box 115, a processing device 116, and a plurality of databases 110 that are communicatively coupled via a network 112.

[0054] Analyzer 108 can be a point-of-care (POC) testing device that performs phenotypic AST of pathogens in patient samples (which can be stored in sample containers 111). In some embodiments, analyzer 108 may be referred to as an analyzer device herein. In some embodiments, the sample in sample container 111 may include whole blood, urine, sterile body fluids or other samples obtained from a patient. In some embodiments, analyzer 108 can receive sample container 111, which is placed in a corresponding drawer in the housing of analyzer 108 by a user or operator of analyzer 108.

[0055] In addition to receiving sample containers 111, the analyzer 108 may also receive sample preparation cartridges 114 and AST cartridges 115, which may similarly be placed by a user or operator of the analyzer 108 into corresponding drawers of the housing of the analyzer 108. In some embodiments, various components and subsystems in the analyzer 108 may interface with the sample containers 111, sample preparation cartridges 114, AST cartridges 115, processing devices 116, and / or databases 110 to perform sample preparation and processing, concentration of pathogens in the sample and subsequent enrichment and cleanup, and AST using microscopy and / or fluorescence.

[0056] In some embodiments, after receiving the sample container 111, the pipetting system in the analyzer 108 can transfer the sample from the sample container 111 to the sample preparation box 114. In some embodiments, the sample preparation box 114 can be a dedicated consumable with a receiving container configured to hold the sample and an element for performing sample processing. The sample preparation box 114 may include a processing tube 113 having a diaphragm disposed on or in the tube for protecting the contents therein. The processing tube 113 can be configured to receive a sample from the sample container 111 through the diaphragm by using a needle of the sample preparation box 114 that transfers the sample. Once the sample is transferred to the processing tube 113, the sample can be subjected to sample concentration, lysis, enrichment, cleaning and / or other processing steps.

[0057] In some embodiments, the sample container 111 is a blood collection tube. Blood collection tubes are available from various suppliers and may include a variety of reagents for preserving blood.

[0058] In some embodiments, sample container 111 is a blood culture bottle. Blood culture bottles are available from different suppliers and contain growth medium that promotes microbial growth. The blood culture bottle may also include a resin that absorbs antibiotics to reduce their effects on the microorganisms in the sample. In some embodiments, the sample in the blood culture bottle is incubated for a certain amount of time to allow pathogens to grow but not reach a growth plateau (positive).

[0059] After sample preparation and processing (e.g., including concentration, enrichment, and cleaning of the sample) using the elements and components of the sample preparation box 114, the sample is transferred from the processing tube 113 in the sample preparation box 114 to the AST box 115 by the pipetting system in the analyzer 108. In some embodiments, the AST box 115 can be a dedicated consumable with multiple reaction wells, which are configured to hold multiple aliquots of the enriched sample for AST. In some embodiments, the analyzer 108 can use microscopy (e.g., single cell microscopy, fluorescence microscopy, etc.) by exposing multiple aliquots of the enriched sample to a predetermined concentration of an antimicrobial agent in the AST box 115.

[0060] In some embodiments, analyzer 108 can be further docked with other boxes (such as polymerase chain reaction (PCR) boxes). In some embodiments, PCR boxes can be special consumables with multiple reaction chambers, and the multiple reaction chambers are configured to hold multiple aliquots of samples for nucleic acid amplification steps for pathogen identification. In some embodiments, analyzer 108 can be configured to hold one or more sample preparation boxes 114, PCR boxes and / or AST boxes 115 at the same time, to simultaneously or continuously perform sample preparation / processing, pathogen identification and / or sensitivity testing. In some embodiments, sample preparation boxes 114, AST boxes 115 and / or PCR boxes can be referred to herein as consumables or containers configured to be inserted into analyzer 108.

[0061] In some embodiments, the analyzer 108 may include a controller 109 disposed inside the housing of the analyzer 108. The controller 109 may control the movement and operation of different components within the analyzer 108, including the movement of one or more sample containers, processing tubes, boxes, and pipetting systems in the analyzer 108. In some embodiments, the controller 109 may also control the operation of one or more centrifuges, subsystems, and modules in the analyzer 108 to perform sample preparation, pathogen identification, sensitivity testing, and / or other functions. In some embodiments, the controller 109 may include a microcontroller on an integrated circuit (IC) chip in the analyzer 108, which is programmed to turn on / off and operate one or more centrifuges, subsystems, and modules in the analyzer 108. In some embodiments, the controller 109 may be coupled to one or more stepper motors, actuators, or other motion control components in the analyzer 108 and programmed to control movement.

[0062] In some embodiments, the controller 109 can be programmed by the processing device 116. The processing device 116 can be a computing device coupled to the analyzer 108 for performing data processing and providing instructions to the controller 109 and / or other components in the analyzer 108. In some embodiments, the processing device 116 can be a personal digital assistant, a desktop workstation, a laptop or notebook computer, a netbook, a tablet computer, a smartphone, a mobile phone, a smart watch, or any combination thereof.

[0063] In some embodiments, the processing device 116 can communicate with the analyzer 108 to receive the results of the reaction occurring in the AST cartridge 115 and perform further processing and data analysis to determine the sensitivity of one or more pathogens of the sample. In some embodiments, the processing device 116 can receive one or more images of the AST cartridge 115 from the analyzer 108 and compare the images to a control to determine whether a particular pathogen is sensitive, intermediately sensitive, or resistant to a particular antimicrobial agent.

[0064] In some embodiments, the processing device 116 can also communicate with multiple databases 110. In some embodiments, one or more of the multiple databases 110 can represent any number of databases, and can include various databases storing clinical parameter data, epidemiological information, or antibiotic resistance information of multiple pathogens. In some embodiments, one or more of the multiple databases 110 can be configured to store pathogen taxonomy data and / or outcomes from a previous pathogen identification workflow (e.g., performed by the analyzer 108). In some embodiments, information from one or more databases 110 can be used to select which antimicrobial agents to test using the AST box 115 and the analyzer 108. In some embodiments, information from one or more databases 110 can be used to determine whether the tested pathogen is resistant, moderately resistant, or sensitive to the drug by the test results obtained by the AST system 101. In some embodiments, one or more of the multiple databases 110 can be configured to store predefined rules for calling the sensitivity of antimicrobial agents for various pathogens. In some embodiments, the processing device 116 can use at least one of the pathogen taxonomy data, the outcome, and / or the predefined rules in the database 110 to report sensitivity information to the user of the analyzer 108. In some embodiments, based on identifying that the pathogen is not sensitive to a particular drug from parsing the predefined rules, the processing device 116 may not report the sensitivity of the pathogen to a particular drug. For example, Klebsiella is naturally resistant to ampicillin, so the processing device 116 may not report this sensitivity information to the user.

[0065] 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 providers, and pharmacies). In some embodiments, the EHR data stored in the database 110 may include patient data and medical history data regarding the health and treatment of the patient, including each patient's demographics, medical history, medications and allergies, immune status, laboratory test results, radiology images, vital signs, personal statistics (like age and weight), and billing information. In some embodiments, the processing device 116 may use the results of pathogen identification and / or AST performed by the analyzer 108, as well as the data stored in the plurality of databases 110 (e.g., clinical parameter data, epidemiological information or antibiotic resistance information, EHR data, etc.) to determine treatment recommendations for the patient.

[0066] In some embodiments, the components in the system 101 can be communicatively coupled via a network 112. In particular, the network 112 can allow information transfer and communication between the analyzer 108, the plurality of databases 110, the processing device 116, and / or any other devices or components in the system 101. In some embodiments, the system 101 can include additional components, such as a Raman spectroscopy device and / or an electronic health record (EHR) system (not shown).

[0067] In some embodiments, the network 112 can be any one or any 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 other suitable configurations. The network can comply with one or more network protocols, including Institute of Electrical and Electronics Engineers (IEEE) protocols, 3rd Generation Partnership Project (3GPP) protocols, 4th generation wireless protocols (4G) (e.g., Long Term Evolution (LTE) standards, advanced LTE, advanced LTE Pro), fifth generation wireless protocols (5G), and / or similar wired and / or wireless protocols, and can include one or more intermediate devices for routing data between the analyzer 108, the plurality of databases 110, the processing device 116, and / or any other devices or components in the system 101. Analyzer device implementation plan:

[0068] Figure 2A diagram of an analyzer device 200 according to an embodiment of the present disclosure is shown. The analyzer device 200 represents Figure 1 108 is an exemplary embodiment of the analyzer 108 shown in . In some embodiments, the analyzer device 200 may be referred to as analyzer 200 herein. The analyzer device 200 is a desktop device having a housing 201, which contains various components and modules for sample preparation, processing and testing. In some embodiments, the housing 201 may include the body of the analyzer device 200 and / or the housing of the analyzer device 200 (which protects the modules and components therein). In some embodiments, the analyzer device 200 may include a cube, a cuboid, or a rectangular shape having various compartments for access and operation by a user of the analyzer device 200. In some embodiments, the analyzer device 200 may have a size of less than about 1 m 3 (eg, compact size of approximately 750 mm (length) x 650 mm (width) x 650 mm (height)).

[0069] In some embodiments, the analyzer device 200 can be coupled to a computing device (e.g., the processing device 116), such as a personal digital assistant, a desktop workstation, a laptop or notebook computer, a netbook, a tablet computer, a smart phone, a mobile phone, a smart watch, or any combination thereof. A user or operator of the analyzer device 200 can use the computing device to control the analyzer device 200, send / receive sample information, patient information, pathogen information, antimicrobial agent information, etc. to / from the analyzer device 200, and access / edit the results of sensitivity testing from the analyzer device 200.

[0070] Figure 3A A diagram showing a front view of an analyzer device 200 according to an embodiment of the present disclosure is shown. Figure 3A The internal features disposed within the housing 201 of the analyzer device 200 are shown, including a first pipette 202, a second pipette 204, a sample drawer 210, a sample cartridge drawer 220, and a processing cartridge drawer 230.

[0071] In some embodiments, the first pipette 202 and the second pipette 204 can be pipette devices configured to manipulate liquid transfer between components within the housing 201 of the analyzer device 200. In some embodiments, the first pipette 202 and the second pipette 204 can be automated devices controlled by the controller 109 in the analyzer device 200. In some embodiments, the controller 109 can control the movement of the first pipette 202 and the second pipette 204, including vertical and / or horizontal movement of the first pipette 202 and the second pipette 204 to and / or from different components within the analyzer device 200. In some embodiments, there can be more or fewer pipettes in the analyzer device 200.

[0072] In some embodiments, the first pipette 202 and the second pipette 204 can be referred to as a high-capacity pipette and a low-capacity pipette, respectively. In some embodiments, the first pipette 202 can be configured to manipulate a volume in the range of about 50 microliters (μL) to 5 milliliters (mL), and the second pipette 204 can be configured to manipulate a volume in the range of about 1 to 200 μL. In some embodiments, the first pipette 202 can have a 5% coefficient of variation (CV) at 50 μL and can have a 1% CV at 5mL, and the second pipette 204 can have a 5% CV at 1 to 200 μL.

[0073] In some embodiments, the first pipette 202 and the second pipette 204 may be referred to herein as a pipette and / or pipette system. In some embodiments, the first pipette 202 and the second pipette 204 may each be configured to be attached to a removable and disposable needle. The first pipette 202 and the second pipette 204 may be attached to two different needles, which are configured to be used to manipulate different volume ranges as necessary for the first pipette 202 and the second pipette 204.

[0074] In addition to liquid transfer, the first pipette 202 and the second pipette 204 can be configured to move elements such as tubes, boxes, etc. within the housing 201 of the analyzer device 200. In particular, the first pipette 202 and the second pipette 204 can each include a pipette tip holder that can be attached to various components used in the analyzer device 200. In particular, the pipette tips of the first pipette 202 and the second pipette 204 can be pressed and fitted into the manipulation features of needles, tubes, boxes, spin column baskets, etc. to pick up various components and place them in different modules or areas in the analyzer device 200.

[0075] In some embodiments, the size of the first pipette 202 and the second pipette 204 can be about 325 mm (width) x 575 mm (depth) x 435 mm (height). In some embodiments, the first pipette 202 and the second pipette 204 can be arranged in the top portion of the housing 201 so that the first pipette 202 and the second pipette 204 can interact with samples, tubes, boxes, and different modules in the bottom portion of the housing 201. In particular, the first pipette 202 and the second pipette 204 can manipulate Figure 3A Liquid transfer and movement of components in sample drawer 210, sample cartridge drawer 220, and process cartridge drawer 230 are shown.

[0076] In some embodiments, the sample drawer 210, the sample box drawer 220, and the processing box drawer 230 include sliding horizontal compartments that are designed to fit into three corresponding receiving containers in the housing 201 of the analyzer device 200. In some embodiments, the sample drawer 210, the sample box drawer 220, and the processing box drawer 230 can be configured to receive special elements for sample processing and testing inserted into the analyzer device 200. In particular, the sample drawer 210 can receive a sample container (e.g., sample container 111) containing a sample obtained from a patient. The sample box drawer 220 can receive a sample preparation box (e.g., sample preparation box 114), into which the sample is transferred by components in the analyzer device 200.

[0077] In some embodiments, the processing box drawer 230 can receive AST boxes (for example, AST boxes 115), and the AST boxes are configured to receive aliquots of enriched samples after processing and enriching samples in the sample preparation box. In other or alternative embodiments, the processing box drawer 230 can be used as AST boxes and / or PCR box drawers. For example, PCR boxes or AST boxes can be inserted in the processing box drawer 230, depending on whether the analyzer device 200 is being used to carry out pathogen identification or antimicrobial susceptibility testing of samples. In some embodiments, the analyzer device 200 can be configured to carry out two functions of pathogen identification and antimicrobial susceptibility testing with dual processing box drawers 230, and the dual processing box drawers are configured to dock with special boxes or consumables for AST and pathogen identification.

[0078] In some embodiments, sample drawer 210, sample box drawer 220 and processing box drawer 230 can each include a reader, and the reader is configured to scan the identifier of sample container, sample preparation box and AST box (or PCR box) respectively. In some embodiments, the reader in the drawer can scan the identifier of sample container and / or box in the housing 201 process of each drawer being inserted into analyzer device 200. In some embodiments, the reader in drawers 210, 220 and 230 can be configured to scan the identification code, bar code or data matrix of corresponding sample container and / or box. In some embodiments, the reader in drawers 210, 220 and 230 can be a bar code reader, quick response (QR) code reader etc.

[0079] In some embodiments, Figure 3A Additional components are shown including one or more centrifuges disposed within housing 201 and configured for manipulating tubes and cartridges for sample processing.

[0080] Figure 3B A diagram showing a top view of an analyzer device 200 according to an embodiment of the present disclosure is shown. Figure 3B A cross section of the analyzer device 200 from a top view is shown, illustrating various components, modules and / or subsystems arranged within the housing 201 of the analyzer device 200 below the first pipette 202 and the second pipette 204 .

[0081] Figure 3B The housing 201 in the embodiment includes a sample drawer 210 for accommodating a plurality of sample containers 212, a sample box drawer 220 for accommodating a plurality of sample preparation boxes 224, and a processing box drawer 230 for accommodating an AST box 232 and a PCR box 234. The sample container 212, the sample preparation box 224, and the AST box 232 represent Figure 1 An exemplary embodiment of a sample container 111 , a sample preparation cartridge 114 , and an AST cartridge 115 is shown.

[0082] In some embodiments, the processing box drawer 230 can be configured to hold both the AST box 232 and the PCR box 234 for pathogen identification and antimicrobial sensitivity testing. In some embodiments, there can be a predefined number of sample containers 212, sample preparation boxes 224, AST boxes 232 and / or PCR boxes 234, which are simultaneously held in their corresponding drawers in the housing 201. In some embodiments, the sample preparation box 224, AST box 232 and / or PCR box 234 can be disposable after a single use or can be reused for testing additional samples.

[0083] Figure 3BAlso shown are 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 .

[0084] In some embodiments, the first centrifuge 240 can be a high-speed centrifuge configured to centrifuge a processing tube (e.g., processing tube 113) and / or a spin column basket placed in the first centrifuge 240 by the first pipette 202. In some embodiments, the second centrifuge 242 can be a low-speed centrifuge configured to centrifuge an AST cartridge 232 placed in the second centrifuge 242 by the second pipette 204. In some embodiments, the first centrifuge 240 and the second centrifuge 242 can centrifuge processing tubes, spin column baskets, and / or AST cartridges 232 in a swing-bucket configuration. In some embodiments, the first centrifuge 240 and the second centrifuge 242 can include a cylindrical shape having a diameter of about 250 mm and a height of about 175 mm.

[0085] In some embodiments, the PCR subsystem 244 may include a thermal cycler and an optical system configured to control temperature when performing quantitative PCR (qPCR), and the optical system is used to perform optical interrogation of the reaction chamber in the PCR box by fluorescence. In some embodiments, the thermal cycler of the PCR subsystem 244 can control the temperature within a range of about 35°C to 100°C. In some embodiments, the optical system of the PCR subsystem 244 can perform fluorescence readings from the bottom of the PCR box in the housing 201. In some embodiments, the PCR box can be placed in the PCR subsystem 244 and removed from the PCR subsystem 244 by the second pipette 204. In some embodiments, the PCR subsystem 244 can hold up to two PCR boxes at a time, wherein the PCR boxes are independently subjected to thermal cycles in the PCR subsystem 244. In some embodiments, the size of the PCR subsystem 244 can be about 70 mm (width) x 125 mm (depth) x 250 mm (height).

[0086] In some embodiments, the enrichment subsystem 246 can be configured to be used for multiple treatment tubes to be maintained in the groove, for applying a rocking motion to the treatment tubes, to allow the oscillation of the sample and the mixing of the pathogen in the growth medium and the sample to carry out the growth and enrichment of the pathogen. In some embodiments, the first pipette 202 can be configured to be used for pipe to be vertically loaded into the groove in the enrichment subsystem 246. In some embodiments, the enrichment subsystem 246 can change the orientation of the pipe from the vertical position to the horizontal position, and apply a rocking motion of ± 15 ° around the horizontal position at a frequency of 1 Hz. In some embodiments, the enrichment subsystem 246 can hold up to about four 15 mL treatment tubes at one time, wherein each treatment tube represents a different sample.

[0087] In some embodiments, the enrichment subsystem 246 can be equipped with a movable magnet. When in a vertical position in the enrichment subsystem 246, the movable magnet can engage the processing tube. In some embodiments, the enrichment subsystem 246 can apply 37°C temperature control to the processing tube maintained in the enrichment subsystem 246. In some embodiments, the size of the enrichment subsystem 246 can be about 150mm (width) x 175mm (depth) x 115mm (height).

[0088] In some embodiments, mechanical device 248 can be configured to stir the treatment tube to carry out the lysis of the microorganism in the sample. In some embodiments, the first pipette 202 can be configured to be used for vertically loading the tube into the groove in the mechanical device 248. In some embodiments, mechanical device 248 can include a stirrer device or a cell disruption device, and the stirring device or the cell disruption device are configured to apply rapid vibration motion to the treatment tube. In some embodiments, mechanical device 248 can apply rapid vibration motion by applying reciprocating motion along a predetermined axis by a stirrer when the treatment tube is in a vertical position.

[0089] In some embodiments, the mechanical device 248 can hold up to two process tubes at a time and apply ±2° of angular motion to the process tubes. In some embodiments, the mechanical device 248 can oscillate the process tubes at about 5,000-30,000 cycles / 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).

[0090] In additional or alternative embodiments, the mechanical device 248 may include a sonicator configured to sonicate the processing tube to agitate the sample.

[0091] In some embodiments, the AST subsystem 250 may include a heater and an imaging subsystem, the heater being configured to incubate samples in the AST box 232 in the housing 201, and the imaging subsystem being used to image the reaction wells of the AST box 232. In some embodiments, the heater of the AST subsystem 250 may be used to control the incubation temperature of the samples in the AST box 232. In some embodiments, the imaging subsystem of the AST subsystem 250 may include a microscope configured to acquire images by scanning the bottom of each reaction well of the AST box 232 using a motorized XYZ translation stage. In some embodiments, the imaging subsystem of the AST subsystem 250 may include a fluorescence sensor, and two optical channels for detecting different fluorescence signals (e.g., green and red).

[0092] In some embodiments, the AST subsystem 250 can identify the antimicrobial phenotypic resistance of the microorganism (e.g., pathogen) based on the acquired image and / or signal. In some embodiments, the AST box 232 can be placed in the AST subsystem 250 and removed from the AST subsystem 250 by the second pipette 204.

[0093] In some embodiments, the AST subsystem 250 can hold up to five AST cassettes 232 at a time. In some embodiments, the AST subsystem 250 can apply C temperature control to the AST cassettes 232 held in the AST subsystem 250, such as by using a thermal block. In some embodiments, the AST subsystem 250 can apply temperature control of about 37°C, and in some embodiments, the dimensions of the AST subsystem 250 can be about 200 mm (width) x 185 mm (depth) x 285 mm (height).

[0094] Figure 4 A diagram of an analyzer device 200 having three open compartments according to an embodiment of the present disclosure is shown. In some embodiments, Figure 41 shows a sample drawer 210, a sample box drawer 220, and a processing box drawer 230 extending from a housing 201 in an open position for loading and / or unloading samples and / or boxes. The sample drawer 210, the sample box drawer 220, and the processing box drawer 230 can be pushed into the housing 201 in a closed position, wherein the compartments fit into three corresponding receiving containers in the housing 201. In some embodiments, the opening and closing of the sample drawer 210, the sample box drawer 220, and the processing box drawer 230 can be controlled by a controller (e.g., controller 109) of the analyzer device 200 and / or by the processing device 116. In some embodiments, a user of the processing device 116 can control the opening and closing of the sample drawer 210, the sample box drawer 220, and the processing box drawer 230 by 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 may 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 analyzer device 200 .

[0095] In some embodiments, sample drawer 210 can hold multiple samples. In some embodiments, sample drawer 210 can hold up to 10 samples at a time, including samples stored in sample containers 212. In some embodiments, Figure 4 The sample container 212 shown can represent one or more blood sample tubes, urine sample tubes and / or blood culture bottles. In some embodiments, the sample box drawer 220 can hold multiple sample preparation boxes at a time, such as up to 10 sample preparation boxes. In some embodiments, the processing box drawer 230 can hold multiple processing boxes at a time, such as up to 12 PCR boxes, 6 AST boxes or a combination thereof.

[0096] In some embodiments, the dimensions of the sample drawer 210 can be about 40 mm (width) x 155 mm (depth) x 600 mm (height). In some embodiments, the dimensions of the sample box drawer 220 can be about 85 mm (width) x 150 mm (depth) x 615 mm (height). In some embodiments, the dimensions of the process box drawer 230 can be about 140 mm (width) x 155 mm (height) x 270 mm (depth). Implementation of sample handling, concentration, enrichment and clean-up in the analyzer device:

[0097] In some embodiments, components disposed within the analyzer device 200 may perform sample processing, concentration, enrichment, and cleanup steps in the processing tube 113, and then run an AST assay and acquire an image of the sample in the AST cartridge 232. In some embodiments, the first pipette 202 of the analyzer device 200 may transfer the sample from the sample container 111 to the processing tube 113, and perform steps to process, separate, concentrate, and enrich pathogens in the sample for rapid detection.

[0098] In some embodiments, sample processing can include the step of blood cell lysis. In the embodiment where the sample is a blood sample, the first pipette 202 can be configured to perform the blood cell lysis of the blood sample. The first pipette 202 can add one or more lysis reagents to the treatment tube 113. The mixer (for example, such as in the enrichment subsystem 246) arranged in the housing 201 of the analyzer device 200 can be used to mix one or more lysis reagents with the blood sample in the treatment tube 117 to make the blood cell lysis in the blood sample. In some embodiments, one or more lysis reagents can include one or more buffers based on saponin. In some embodiments, one or more lysis reagents can include one or more detergents, surfactants or proteases.

[0099] After the blood cells in the blood sample are lysed, the first pipette 202 can transfer the processing tube 117 to the centrifuge 240 (or Fig.14A 1402) to concentrate and enrich the lysed sample. Concentration and enrichment of the sample can involve a series of steps using components in the analyzer device 200, including centrifugation. In some embodiments, the first pipette 202 in the analyzer device 200 can move the processing tube 117 to a centrifuge, such as the first centrifuge 240 or 1402. In some embodiments, the centrifuge 240 or 1402 can apply centrifugal force to the processing tube 117 to concentrate the pathogens in the sample, such as by separating the pathogens from other components in the sample.

[0100] After centrifugation, the first pipette 202 can be used to remove fluid from the processing tube 117, leaving concentrated pathogens in the processing tube 117. In some embodiments, the first pipette 202 can then add growth medium to the concentrated pathogens in the processing tube 117 so that the concentrated pathogens grow in the processing tube 117 for a predetermined period of time. In some embodiments, the predetermined period of time can be a period of time that allows the concentrated pathogens to grow, such as about 4 hours or longer. In some embodiments, for the enrichment of most pathogens, the predetermined period of time can be 3-4 hours, while other pathogens may require a longer period of time for growth / enrichment. In some embodiments, the growth medium can be stored in one or more reservoirs of the sample preparation box 224. In some embodiments, the growth medium can include Mueller Hinton broth, cation-adjusted Mueller Hinton broth, trypticase soy broth, lysogeny broth, brain heart infusion (BHI) broth, etc.

[0101] After concentration and enrichment, the analyzer device 200 can perform a cleaning or cleanup step of the sample to obtain the pathogens in the enriched sample. In some embodiments, to clean the concentrated pathogens, the first pipette 202 can move the processing tube 117 to the centrifuge 240 or 1402 after a predetermined period of growth has passed (after adding the growth medium), and remove the supernatant from the processing tube 117 to obtain the enriched sample.

[0102] In some embodiments, the analyzer device 200 can achieve the clean-up step of the sample by immunomagnetic separation (IMS) technology (such as by using magnetic beads). In some embodiments, the processing tube 117 can include a plurality of magnetic beads configured to attach to the concentrated pathogens in the processing tube 117. In some embodiments, the magnetic beads can be coated with non-specific ligands. In some embodiments, the magnetic beads are coated with specific ligands that are specific to a specific pathogen or group of pathogens of the concentrated pathogens in the processing tube 117. In some embodiments, the pathogens can be fixed on the surface of the magnetic beads after an incubation period and can be concentrated into pellets using a magnetic field.

[0103] In some embodiments, the first pipette 202 can move the processing tube 117 to a magnet station (such as in the enrichment subsystem 246) to apply a magnetic force to the processing tube 117 to retain the concentrated pathogens attached to the magnetic beads. In some embodiments, after retaining the concentrated pathogens attached to the magnetic beads in the processing tube 117, the first pipette 202 can then remove the extraneous liquid from the processing tube 117. Removal of the extraneous liquid can produce an enriched sample with concentrated pathogens.

[0104] In some embodiments, the concentrated pathogens can be resuspended and washed to remove any debris or other materials in the washing step. In particular, the first pipette 202 can add one or more washing materials to the concentrated pathogens in the processing tube 117 to clean and remove any blood components or debris from the concentrated pathogens, leaving an enriched sample in the processing tube 117. In some embodiments, the one or more washing materials can include a combination of one or more buffers, detergents, surfactants, and proteases. In some embodiments, the one or more washing materials can be stored in the sample preparation box 224.

[0105] In some embodiments, analyzer 200 can be configured to identify multiple pathogens in a sample. In particular, analyzer 200 can use one or more fluorescent dyes and a microscope (e.g., in AST subsystem 250) to mark cells in a sample and count the number of pathogens. In some embodiments, the number of pathogens in an initial sample (e.g., before concentration and enrichment) can be less than 200. By performing concentration and enrichment steps, the number of pathogens in the enriched sample can be increased to a range of about 1,000 to about 100,000. In some embodiments, the number of pathogens in the enriched sample can be about 10,000. In response to the identification of the number of pathogens in the sample, analyzer 200 can be configured to further enrich or dilute the sample to obtain a predetermined number of pathogens in the enriched sample.

[0106] In some embodiments, the sample can be treated with protease and / or DNA enzyme before or after concentration. For example, protease and / or DNA enzyme can be added to the sample after concentration but before enrichment. In another example, protease and / or DNA enzyme can be added to the sample simultaneously with a cleavage reagent.

[0107] In some embodiments, sample can be processed with protease and / or DNA enzyme during the cleaning process. For example, protease and / or DNA enzyme can be added during the immunomagnetic capture process to digest and reduce the amount of fragments in the sample. In another example, protease and / or DNA enzyme can be added after the immunomagnetic capture process, which has the advantage of avoiding any interference of protease / DNA enzyme reagent to the IMS process. When the capture of the bacterium by the pearl is based on the protein that may be degraded by protease and hinder its effect, this is particularly important.

[0108] After concentration, enrichment, and cleanup, the enriched sample in the processing tube 117 may then be ready to be transferred to the AST cartridge 232 via a pipetting system in the analyzer 200 for AST assay and image acquisition to determine the sensitivity of the concentrated pathogens in the enriched sample. Sample preparation kit implementation plan:

[0109] Figure 5 A diagram of a sample preparation kit 500 according to an embodiment of the present disclosure is shown. The sample preparation kit 500 represents Figure 3B 224. The sample preparation box 500 can be a consumable inserted into the sample drawer 210 of the analyzer device 200 for preparing and processing samples in the sample container. In some embodiments, the sample preparation box 500 can be made of a polypropylene (PP) material by injection molding. The sample preparation box 500 can include a housing 502, a plurality of reservoirs 504, a cover 506, and an identifier 508.

[0110] In some embodiments, the housing 502 may have an elongated rectangular shape with rounded edges. The housing 502 may be configured to hold a Fig. 6A In some embodiments, a plurality of reservoirs 504 can be separate reservoirs or tubes molded together. A plurality of reservoirs 504 can be configured to store materials for carrying out sample concentration, cracking and / or nucleic acid amplification. In some embodiments, the material stored in the reservoir 504 can include one or more buffers (for example, buffers based on NaCl, phosphate buffered saline (PBS, etc.), detergents, surfactants, proteases, growth medium, etc. In some embodiments, the reservoir 504 can store one or more lysis reagents, such as one or more buffers based on saponin, detergents, surfactants or proteases, for carrying out the cell lysis of a blood sample. In some embodiments, the reservoir 504 can store one or more washing materials, and the one or more washing materials can include a combination of one or more buffers, detergents, surfactants and proteases.

[0111] In some embodiments, the cover 506 of the sample preparation box 500 is a protective cover that extends across and covers the housing 502 and the plurality of reservoirs 504. In some embodiments, the identifier 508 is an identifier of the sample preparation box 500 that can be scanned by the analyzer device 200 to perform sample preparation for antimicrobial susceptibility testing. In some embodiments, the identifier 508 can be at least one of an identification code, a bar code, or a data matrix (such as a QR code). In some embodiments, the size of the sample preparation box 500 can be as follows: Figure 5 Approximately 80mm (width) x 55mm (depth) x 155mm (height) as shown.

[0112] Fig. 6AA diagram showing an exploded view of a sample preparation cartridge 500 having a processing tube 510 and other components to be inserted therein according to an embodiment of the present disclosure is shown. The sample preparation cartridge 500 may include a processing tube 510, a first removable needle 512, and two second removable needles 514, which are stored in corresponding receiving containers in a housing 502 of the sample preparation cartridge 500. The processing tube 510 represents Figure 1 An exemplary embodiment of a process tube 113 is shown.

[0113] In some embodiments, the processing tube 510 may include, for example, a 15 ml tube with a conical bottom. In some embodiments, the processing tube 510 may be removed from the sample preparation box 500 for processing in other modules in the analyzer device 200. In some embodiments, the processing tube 510 may be a tube into which the sample is transferred after the sample container and the sample preparation box 500 are loaded into the sample drawer 210 and the sample box drawer 220 of the analyzer device 200, respectively.

[0114] In some embodiments, a sample can be transferred from a sample container in a sample drawer 210 to a processing tube 510 of a sample preparation cartridge 500 in a sample cartridge drawer 220 via a first removable needle 512. In some embodiments, a first pipette 202 can be attached with the first removable needle 512, which is configured for transferring a sample into and / or from a 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 can be used to manipulate small volumes, and the second pipette 204 can be configured for attachment with the two second removable needles 514.

[0115] Fig. 6A Also shown are openings 520 of the plurality of reservoirs 504 of the sample preparation cartridge 500. In some embodiments, there may be 12 reservoirs 504 in the sample preparation cartridge 500, wherein each reservoir 504 holds a volume of approximately 2.5 mL. In some embodiments, the sample preparation cartridge 500 may include a pierceable membrane 516 covering the receiving container 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 membrane 516 may be a polyester film, and the foil seal 518 may include an aluminum foil, wherein both the pierceable membrane 516 and the foil seal 518 may be pierced by the first removable needle 512 and / or the second removable needle 514.

[0116] Figure 6BA diagram showing an exploded view of a sample preparation cartridge 500' having a processing tube 510 and other components to be inserted therein according to an embodiment of the present disclosure. Sample preparation cartridge 500' is similar to sample preparation cartridge 500, but has a more linear form factor and a peelable film 506' instead of lid 506. Processing tube and needle implementation:

[0117] Fig. 7A and Figure 7B A diagram of a process tube 700 is shown according to an embodiment of the present disclosure. The process tube 700 represents Fig. 6A An exemplary embodiment of a processing tube 510 is shown. In particular, Fig. 7A The assembled process tube 700 is shown, and Figure 7B An exploded view of the components in process tube 700 is shown. Process tube 700 includes cap 702, septum 704, and tube 706.

[0118] In some embodiments, the septum 704 can be affixed to the interior of the tube 706, with the cap 702 fitting over the septum 704 of the processing tube 700. In some embodiments, the septum 704 can be configured for insertion and removal of a needle (e.g., needles 512 and 514) to transfer liquid to and from the processing tube 700 without removing the cap 702 from the tube 706. In some embodiments, the septum 704 can provide an airtight seal within the processing tube 700 and prevent contamination of the contents of the processing tube 700.

[0119] In some embodiments, the processing tube 700 can include a handling feature at the top of the processing tube 700 that is compatible with being manipulated by a pipette (e.g., the first pipette 202 and / or the second pipette 204). In some embodiments, the handling feature of the processing tube 700 can be a cylindrical cavity in the cap 702 that is compatible with being inserted by the tip of the pipette. In some embodiments, the pipette tips of the first pipette 202 and the second pipette 204 can be pressed and fit into the cylindrical cavity in the cap 702 to pick up and move the processing tube around the analyzer device 200.

[0120] In some embodiments, cap 702 can be made of high density polyethylene (HDPE) material, and tube 706 can be made of polypropylene (PP) material. In some embodiments, diaphragm 704 can include at least one of rubber, polytetrafluoroethylene (PTFE), thermoplastic elastomer (TPE), silicone, butyl rubber or its combination. In some embodiments, diaphragm 704 can include a double layer of polytetrafluoroethylene (PTFE) and another material, and the another material is selected from silicone, rubber and butyl rubber. In some embodiments, the size of treatment tube 700 after assembly can include, for example, a height of about 110mm and a diameter of about 20mm. In some embodiments, diaphragm 704 can have a thickness ranging from about 1 to 2mm.

[0121] In some embodiments, the processing tube 700 can 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 can be coated with a non-specific ligand. In some embodiments, the plurality of magnetic beads can be coated with a specific ligand that is specific for a particular pathogen of the concentrated pathogens in the processing tube 700. In additional or alternative embodiments, the plurality of magnetic beads can be stored in one or more reservoirs 504 of the sample preparation cartridge 500.

[0122] In some embodiments, the processing tube 700 can be configured to receive Fig. 8A , Figure 8B and Figure 8C The needle shown. Fig. 8A , Figure 8B and Figure 8C A diagram of a needle 800 configured for insertion into a processing tube 700 is shown, according to some embodiments of the present disclosure.

[0123] in particular, Fig. 8AA needle 800 including a plastic body 810, a cannula 820, and a plurality of grooves 825 is shown. In some embodiments, the plastic body 810 can be attached to the cannula 820 by bonding. In some embodiments, one or more pipettes (e.g., the first pipette 202) in the analyzer device can be attached to the 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 pipettes in the analyzer device. In some embodiments, the needle 800 can be a venting needle configured to ventilate the processing tube 700 when the needle is inserted. In some embodiments, the ventilation of the needle 800 can help release the pressure in the sealed processing tube 700. In some embodiments, the plastic body 810 of the needle 800 includes a predetermined number of grooves 825, which are configured to provide an air connection between the inside and the outside of the processing tube 706 when the needle 800 is inserted through the septum 704 and inserted into the tube 700. For example, there may be four grooves 825 in the injection portion of the retaining sleeve 820 of the needle 800. In some embodiments, the grooves 825 may be produced by injection molding.

[0124] Figure 8B The needle 800 is shown during insertion into the processing tube 700, and Figure 8C The needle 800 is shown after being fully inserted into the treatment tube 700. In some embodiments, the cannula 820 of the needle 800 can be inserted into the cap 702, through the septum 704, and into the tube 706 of the treatment tube 700. In some embodiments, when the cannula 820 is fully inserted into the tube 706, the distal end of the plastic body 810 can fit into the cap 702 of the treatment tube 700.

[0125] Fig. 9A and Fig. 9B A diagram showing a high volume needle 900 and a low volume needle 910 according to an embodiment of the present disclosure is shown, respectively. In some embodiments, the high volume needle 900 and the low volume needle 910 can be coupled to the first pipette 202 and the second pipette 204 in the analyzer device 200, respectively.

[0126] like Fig. 9A As shown, the high-capacity needle 900 can include a plastic body 902 and a cannula 904. In some embodiments, the plastic body 902 can be a reservoir configured to hold a volume of about 50 μL to 5 mL during liquid transfer in an analyzer device. In some embodiments, the plastic body 902 can include a filter 903 disposed therein to prevent contamination of a pipette coupled to the high-capacity needle 900.

[0127] In some embodiments, the plastic body 902 can be made of a polypropylene (PP) material. In some embodiments, the plastic body 902 of the needle 900 can have a diameter of about 16 mm and a length of about 50 mm. In some embodiments, the cannula 904 can be made of stainless steel. In some embodiments, the cannula 904 can have a length of about 100 mm. In some embodiments, the plastic body 902 and the cannula 904 can have a length of about 150 mm when assembled together. In some embodiments, the high capacity needle 900 can 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.

[0128] In some embodiments, the cannula 904 can further include a secondary cannula disposed around the inner core of the needle 900. In some embodiments, the cannula 904 includes one or more vents 906. In some embodiments, the cannula 904 of the needle 900 can be in fluid communication with the vents 906. In some embodiments, the cannula 904 can be a slotted cannula having slots around the needle.

[0129] like Fig. 9B As shown, the low-capacity needle 910 may include a plastic body 912 and a needle valve stem 914. In some embodiments, the plastic body 912 may be a reservoir configured to maintain a volume of about 1 to 200 μL during liquid transfer in an analyzer device. In some embodiments, the plastic body 912 may include a filter 913 arranged therein to prevent contamination of a pipette coupled to the low-capacity needle 910. In some embodiments, the plastic body 912 may be made of 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 valve stem 914 may have a length of about 10 mm. In some embodiments, the needle valve stem 914 may be made of stainless steel. In some embodiments, the low-capacity 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.

[0130] Fig.10 A diagram showing an example of a high capacity needle 900 according to an embodiment of the present disclosure is shown. In particular, Fig.10 Various examples of cannulas for high capacity needles 900 having vents, slots, etc. are shown. In some embodiments, Fig.10 The first needle in the needle can be vented by having two connected orifices or vents in the cannula of the needle. In some embodiments, Fig.10 The second and fourth needles shown in FIG. 7 can be vented when the tip of each needle is inserted into the septum (eg, septum 704) of the processing tube.

[0131] Fig.11 A diagram of a low volume needle 910 docked with a sample preparation cartridge 500 is shown according to an embodiment of the present disclosure. In particular, Fig.11 An example of a low volume needle 910 disposed in one of the plurality of reservoirs 504 of the sample preparation cartridge 500 is shown, such as for transferring material for sample concentration and / or lysis from the reservoir 504 to a sample in a processing tube. In some embodiments, the sample preparation cartridge 500 can be configured to receive one needle, such as the low volume needle 910. In other embodiments, the sample preparation cartridge 500 can be configured to receive two needles, such as both the high volume needle 900 and the low volume needle 910. AST box implementation plan:

[0132] Fig. 12A and Fig. 12B A diagram of an AST box 1200 according to an embodiment of the present disclosure is shown. The AST box 1200 represents an exemplary embodiment of the AST box 232. In particular, Fig. 12A shows the assembled AST box 1200, and Fig. 12B An exploded view of the components in the AST box 1200 is shown. The AST box 1200 includes a cover 1202, a septum 1208, and a base 1212. In some embodiments, the cover 1202 is disposed on the septum 1208, and the septum is disposed on the base 1212.

[0133] 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 is in the range of about 50 to 200, for example, 100 reaction wells. In some embodiments, each reaction well 1214 can maintain a volume ranging from about 20 to 50 μL, for example, 30 μL. Each reaction well 1214 in the plurality of reaction wells 1214 can accommodate an antimicrobial agent of a predetermined concentration for reacting with a corresponding aliquot of an enriched sample containing a pathogen. In some embodiments, the antimicrobial agent arranged in each reaction well 1214 can be in liquid form or in a dry form or a freeze-dried form. In some embodiments, the antimicrobial agent arranged in each reaction well 1214 can be added to each reaction well 1214 in the base 1212 by a first pipette 202 or a second pipette 204, and then a plurality of aliquots of the enriched sample are distributed to the reaction well 1214. In some embodiments, the antimicrobial agent disposed within each reaction well 1214 can be added to each reaction well 1214 during manufacture and / or assembly of the AST box 1200.

[0134] In some embodiments, after concentrating and enriching the pathogens in the sample in the processing tube 700, the first pipette 202 or the second pipette 204 in the analyzer device 200 can distribute multiple aliquots of the enriched sample into the reaction wells 1214 in the AST box 1200. In some embodiments, each reaction well 1214 can be configured to receive an aliquot of the enriched sample containing the pathogen by non-contact distribution of the aliquot through a needle (e.g., a needle 900 or 910 coupled to the first pipette 202 or the second pipette 204). In some embodiments, the needle can penetrate the diaphragm 1208 of each reaction well 1214 without the needle contacting the bottom surface of each reaction well 1214 to distribute each aliquot. In some embodiments, the volume of each aliquot distributed by the needle 900 or 910 can be in the range of about 0.5 μL to about 10 μL.

[0135] 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 can have a diameter of less than about 2 mm. In some embodiments, non-contact dispensing of multiple aliquots to each reaction well 1214 can include non-contact dispensing of aliquots using jet dispensing through a needle 900 or 910 in the sample preparation box 500, the needle penetrating the corresponding septum of the reaction well 1214 and the needle 900 or 910 not contacting the bottom wall of each reaction well 1214.

[0136] In some embodiments, the bottom wall of each reaction well 1214 can be optically transparent and configured for optical interrogation. In some embodiments, the bottom wall of each reaction well 1214 can be configured for optical interrogation, such as by an AST subsystem 250 in the analyzer device 200. In some embodiments, the bottom wall of each reaction well 1214 can be configured for fluorescence microscopy, such as by an AST subsystem 250 in the analyzer device 200. In some embodiments, a plurality of reaction wells 1214 can be configured to fit into corresponding wells in a temperature control block in the AST subsystem 250, and the temperature control block can be configured to heat the sides of the plurality of reaction wells 1214.

[0137] The diaphragm 1208 seals each of the multiple reaction wells 1214 of the base 1212. In some embodiments, the diaphragm 1208 can be referred to as a sealing cap. In some embodiments, the diaphragm 1208 can include a single body (unibody) that extends across the multiple reaction wells 1214 of the base 1212. In some embodiments, the diaphragm 1208 can include a plurality of parts assembled together, each of which covers each reaction well 1214 of the base 1212. The diaphragm 1208 can be configured to receive a needle, such as a needle 900 or 910 coupled to the first pipette 202 or the second pipette 204. In some embodiments, the needle can create an orifice in the diaphragm 1208 during insertion, and the orifice in the diaphragm 1208 can be closed due to the material of the diaphragm 1208 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 a combination thereof. In some embodiments, the diaphragm 1208 may include a double layer of polytetrafluoroethylene (PTFE) and another material selected from silicone, rubber, and butyl rubber. In some embodiments, the diaphragm 1208 may have a thickness in the range of about 1 to 2 mm.

[0138] In some embodiments, the cover 1202 may include a plurality of openings 1206, wherein each opening 1206 is aligned with a corresponding reaction well 1214 of a 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, which may be scanned by the analyzer device 200 for antimicrobial susceptibility testing. In some embodiments, the identifier 1210 may be at least one of an identification code, a barcode, or a data matrix.

[0139] In some embodiments, the cover 1202 can be fitted onto the diaphragm 1208 and the base 1212 to form the assembled AST box 1200. In some embodiments, the diaphragm 1208 can be overmolded into the cover 1202 to form a combined component, and the combined component can be assembled onto the base 1212 by at least one of a snap-fit ​​joint or a mechanical fastener. In some embodiments, the cover 1202, the diaphragm 1208, and the base 1212 can be interlocked or clamped together by one or more mechanical fasteners.

[0140] In some embodiments, the base 1212, the septum 1208, and the 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 compatible with insertion of a pipette tip (e.g., a first pipette 202 and a second pipette 204) to move the AST cartridge 1200 in the analyzer device 200. In some embodiments, the openings 1204 in the base 1212, the septum 1208, and the cover 1202 may be aligned with each other when the AST cartridge 1200 is assembled.

[0141] In some embodiments, the base 1212 can be made of a polystyrene (PS) material. In some embodiments, the cover 1202 can be made of a polypropylene (PP) or polycarbonate (PC) material. In some embodiments, the size of the assembled AST box 1200 can be about 135 mm (length) x 35 mm (width) x 10 mm (height).

[0142] Fig.13 A diagram of a low volume needle 910 inserted into an AST cartridge 1200 according to an embodiment of the present disclosure is shown. In particular, Fig.13 The needle stem 914 of the needle 910 is shown piercing the septum 1208 and entering the reaction well 1214 of the AST cartridge 1200. In some embodiments, the needle stem 914 can create an orifice in the septum 1208 during insertion. When the needle stem 914 is removed, the orifice in the septum 1208 can close due to the material of the septum 1208. Implementation of modules and subsystems in the analyzer:

[0143] Fig.14A and Fig. 14B A diagram of an exemplary centrifuge for use in an analyzer device 200 according to an embodiment of the present disclosure is shown. In particular, Fig.14A A first centrifuge 1402 is shown that can be used to centrifuge a sample in a tube 1404, and Fig. 14B A second centrifuge 1412 is shown that can be used to centrifuge the enriched sample in the AST cartridge 1414. The first centrifuge 1402 and the second centrifuge 1412 are respectively Figure 3B 1404 and AST box 1414 represent the exemplary embodiment of the first centrifuge 240 and the second centrifuge 242 shown in FIG. Figure 7A-7B and Figure 12A-12B An exemplary embodiment of a processing tube 700 and an AST box 1200 is shown in FIG.

[0144] In some embodiments, the first centrifuge 1402 can be a high-speed centrifuge configured to apply a relative centrifugal force (RCF) or g-force of about 12,000 G to the processing tube 1404. In some embodiments, the second centrifuge 1412 can be a low-speed centrifuge configured to apply a relative centrifugal force (RCF) or g-force of about 3,000 G to the AST box 1414.

[0145] In some embodiments, the first centrifuge 1402 can hold the process tube 1404 in a first orientation and apply a 45° swing bucket centrifugation to the process tube 1404. In some embodiments, the second centrifuge 1412 can hold the AST cartridge 1414 in another orientation and apply a 90° swing bucket centrifugation such that the AST cartridge 1414 moves to a vertical position in the second centrifuge 1412.

[0146] In some embodiments, the first centrifuge 1402 and the second centrifuge 1412 can centrifuge multiple processing tubes 1404 and AST boxes 1414 at a time. For example, the first centrifuge 1402 can be configured to hold two processing tubes 1404 at a time for centrifugation together. In another example, the second centrifuge 1412 can be configured to hold two AST boxes 1414 at a time for centrifugation together. In some embodiments, the processing tube 1404 can be moved to the first centrifuge 1402 during the concentration and enrichment steps to obtain an enriched sample with pathogens.

[0147] Fig.15A , Fig. 15B , Fig. 15C and Fig.15D A diagram of an exemplary enrichment subsystem 1500 for use in an analyzer device 200 according to an embodiment of the present disclosure is shown. The enrichment subsystem 1500 represents Figure 3B246. In some embodiments, the enrichment subsystem 1500 can apply a rocking motion to the treatment tube 700 to allow the sample to oscillate and mix with other materials. In some embodiments, the treatment tube 700 can be placed in the enrichment subsystem 1500 by a pipetting system (e.g., a first pipette 202 or a second pipette 204) for mixing the pathogen in the treatment tube 700 with a growth medium for the enrichment of the pathogen growth and sample. In some embodiments, the treatment tube 700 can be placed in the enrichment subsystem 1500 by a pipetting system (e.g., a first pipette 202 or a second pipette 204) for mixing one or more lysis reagents with the blood sample in the treatment tube 700 to lyse the blood cells in the blood sample. In some embodiments, the mixing function of the enrichment subsystem 1500 can be used for sample processing to perform both pathogen identification and / or antimicrobial sensitivity testing in the analyzer device 200.

[0148] In some embodiments, the enrichment subsystem 1500 can be a mixer so that it rotates one or more treatment tubes 700 in a horizontal position by rocking the treatment tubes 700 back and forth at an angle of ±30°. In some embodiments, four treatment tubes 700 can be loaded into the enrichment subsystem 1500 at a time. In some embodiments, the enrichment subsystem 1500 can include a magnet 1501 that moves back and forth between an upward position and a downward position. In some embodiments, one or more treatment tubes 700 can include magnetic beads configured to attach to concentrated pathogens in the one or more treatment tubes 700. In some embodiments, the magnet 1501 can be used to retain the concentrated pathogens attached to the magnetic beads in the one or more treatment tubes 700.

[0149] In some embodiments, the enrichment subsystem 1500 may include an additional or alternative independent magnet station that can be used to retain the concentrated 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 can 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 to acquire an image of the AST cartridge 1200. In some embodiments, the pathogens in each aliquot can be attached to magnetic beads that allow the pathogens to move to the bottom wall of the reaction well 1214.

[0150] Fig.16A , Fig. 16B and Fig. 16C A diagram of an exemplary mechanical device 1600 for use in an analyzer apparatus 200 according to an embodiment of the present disclosure is shown. The mechanical device 1500 represents Figure 3B. In some embodiments, the mechanical device 1600 can hold two processing tubes 700 in a vertical position and provide rapid vibratory movement to the processing tubes 700, such as for 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 a sonicator configured to sonicate the processing tubes 700 to agitate the sample.

[0151] Fig.17A and Fig. 17B A diagram showing an AST cassette 1200 interfaced with an AST subsystem 1700 in an analyzer according to an embodiment of the present disclosure. The AST subsystem 1700 represents Figure 3B 1700. 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 platform 1706, and a heat block 1708.

[0152] In some embodiments, the AST cartridge 1200 can be placed in the heat block 1708 of the AST subsystem 1700 by a pipette (e.g., the first pipette 202 or the second pipette 204) in the analyzer device 200. In some embodiments, the heat block 1708 can surround all sides of the reaction well 1214 of the AST cartridge 1200 when the AST cartridge 1200 is positioned inside. In some embodiments, the heat block 1708 can apply 37°C temperature control to the AST cartridge 1200. In some embodiments, when positioned in the heat block 1708 of the AST subsystem 1700, a heated cover 1710 can be placed on the AST cartridge 1200. The microscope 1702 can be configured to obtain an optical reading of the bottom wall of each reaction well 1214 in the AST cartridge 1200 through the heat block 1708.

[0153] In some embodiments, the scanning platform 1706 may include a motorized XYZ translation stage that allows the motorized positioning of the microscope 1702 on the AST box 1200. In some embodiments, the microscope 1702 can be configured to scan all reaction holes 1214 in the AST box 1200 at one or more wavelengths (e.g., at two wavelengths) in less than about 300 seconds. In some embodiments, the microscope 1702 can use a 10X objective lens for optical reading. In some embodiments, the microscope 1702 can use two optical channels for detecting different fluorescent signals (e.g., green and red fluorescence). In some embodiments, the microscope 1702 can use two different wavelengths to scan the AST box, such as a 490nm wavelength for excitation and a 520nm wavelength for emission (e.g., for detecting green fluorescence) and a 540nm wavelength for excitation and a 620nm wavelength for emission (e.g., for detecting red fluorescence).

[0154] In some embodiments, the microscope 1702 can be configured to perform fluorescence microscopy of the reaction wells 1214 in the AST cartridge 1200. In some embodiments, before acquiring one or more images using the microscope 1702, the first pipette 202 or the second pipette 204 can dispense the first fluorescent dye and / or the second fluorescent dye into each reaction well 1214 in the AST cartridge 1200 to stain the pathogens in the aliquot. In some embodiments, after incubating the aliquot of the enriched sample in the reaction wells 1214 of the AST cartridge 1200, the first fluorescent dye and / or the second fluorescent dye can be delivered to the reaction wells 1214. In some embodiments, the first fluorescent dye can include a DNA binding dye that marks live cells in the aliquot of the reaction wells 1214, while the second fluorescent dye can include a fluorescent intercalator that cannot cross intact cell membranes and thus only marks dead cells with compromised membranes in the aliquot of the reaction wells 1214. For example, the first fluorescent dye can be Green or another fluorescent dye that emits green fluorescence (e.g., at an emission wavelength of about 500 to 560 nm), and the second fluorescent dye can be propidium iodide (PI) or another fluorescent agent that emits red fluorescence (e.g., at an emission wavelength of about 560 to 700 nm).

[0155] In some embodiments, the first pipette 202 or the second pipette 204 can use jet dispensing to perform non-contact dispensing through the first needle 512 or the second needle 514 (coupled to the first pipette 202 or the second pipette 204) to avoid cross-contamination of the reaction wells of the first fluorescent dye and / or the second fluorescent dye, wherein the first needle 512 or the second needle 514 penetrates the corresponding septum 1208 of the reaction well 1214, and the first needle 512 or the second needle 514 does not contact the bottom wall of each reaction well 1214. In some embodiments, the volume of the first fluorescent dye and / or the second fluorescent dye dispensed to each reaction well 1214 by the first needle 512 or the second needle 514 can be in the range of about 0.5 microliters to 10 microliters. In some embodiments, the microscope 1702 can perform fluorescence microscopy to perform image acquisition for the AST box 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 box 1200. In some embodiments, the microscope 1702 can be configured to detect one fluorescent dye, two fluorescent dyes, etc. to analyze the fluorescent image of the AST box 1200.

[0156] In some embodiments, the microscope 1702 can be coupled to a processor (e.g., processing device 116) configured to perform image analysis and data processing on one or more images obtained from the AST box 1200. In some embodiments, the processor coupled to the microscope 1702 can analyze one or more images obtained from the AST box 1200 by calculating the number of fluorescent pathogens in each reaction well 1214 in the AST box 1200. In some embodiments, the processor can determine whether the pathogens in the sample are resistant to various antimicrobial agents based on calculating the number of live cells and the number of dead cells in the reaction wells 1214 of the AST box 1200 and applying various rules. For example, the processor can identify whether a specific 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 lower than a predetermined threshold value of a predetermined antimicrobial concentration.

[0157] In another example, the processor can identify whether a specific pathogen is resistant by determining whether the ratio of the number of dead cells to the number of live cells is higher than a predetermined threshold for an aliquot of an enriched sample that does not contain an antimicrobial agent in the reaction well 1214. In another example, the processor can identify whether a specific pathogen is resistant by determining whether the ratio of the number of live cells in an aliquot incubated with an antibiotic to the number of live cells in an aliquot not incubated with an antibiotic is higher than a predetermined threshold. In yet another example, the processor can identify whether a specific pathogen is resistant by determining whether the ratio of the number of live cells in an aliquot incubated with an antibiotic to the number of live cells in an aliquot at time zero (e.g., before incubation / reaction) is higher than a predetermined threshold. In yet another example, the processor can identify whether a specific pathogen is resistant by determining whether the ratio of the brightness of live cells in one or more images of an aliquot incubated with an antibiotic to the brightness of live cells in one or more images of an aliquot at time zero (e.g., before incubation / reaction) is higher than a predetermined threshold. In some embodiments, the processor can 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 certain antimicrobial agents. In some embodiments, the microscope 1702 and the processor can acquire and process images of reaction wells 1214, wherein several replicates of each reaction (e.g., between an aliquot of an enriched sample and an antimicrobial agent) are performed in multiple reaction wells 1214 in the AST box 1200.

[0158] In some embodiments, the processor can determine the minimum inhibitory concentration (MIC) of the antibiotic or antimicrobial agent that inhibits the growth of a specific pathogen based on one or more images analyzing AST box 1200. In some embodiments, different aliquots can be used to incubate pathogens under different antibiotic concentrations. In some embodiments, the processor can determine MIC as the minimum concentration of antibiotic, at which, relative to the initial quantity of pathogen, the reaction does not show significant growth. In some embodiments, the processor can determine MIC by the result of extrapolating the intermediate antimicrobial concentration. In some embodiments, the processor can use the value obtained for MIC to determine whether the pathogen is resistant, medium resistant or sensitive based on a lookup table or a logical rule set stored in a database (e.g., database 110). Exemplary operation method:

[0159] Fig.18A flow chart of a method 1800 for performing AST of a sample according to an embodiment of the present disclosure is shown. In some embodiments, the method 1800 may describe steps for performing AST using various components in an AST system, including an analyzer 108, 200; a sample preparation cartridge 114, 224, 500; a processing tube 113, 510, 700; an AST cartridge 115, 232, 1200; a controller 109 and a processing device 116, as described above with reference to Figure 1-Figure 1 7. It should be understood that the operations shown in method 1800 are not exhaustive, and other operations may also be performed before, after, or between any of the operations shown. In various embodiments of the present disclosure, the operations of method 1800 may be performed in different orders and / or variations.

[0160] Fig.18 The method 1800 of the present invention begins at step 1802, in which a sample preparation box and a sample container are received in an analyzer device. In some embodiments, the analyzer device 200 can receive a sample preparation box 224 and a sample container, which are placed in a sample box drawer 220 and a sample drawer 210 of the analyzer device 200 by a user or operator of the analyzer device 200, respectively. In some embodiments, the sample container may include a sample obtained from a patient, wherein the sample contains a pathogen. In some embodiments, the sample in the sample container may include whole blood, urine, sterile body fluids, or other samples obtained from a patient.

[0161] In step 1804, the first needle from the sample preparation cartridge is installed in the pipette system in the analyzer device. In some embodiments, the needle 512 or 514 from the sample preparation cartridge 500 can be installed in the first pipette 202 or the second pipette 204 by moving the pipette tip of the first pipette 202 or the second pipette 204 over the sample preparation cartridge 500, pressing and fitting into the plastic body portion of the needle (e.g., plastic body 902 or 912).

[0162] In step 1806, the first needle is inserted into the sample container using the pipette system. In some embodiments, after the needle 512 or 514 is installed, the first pipette 202 or the second pipette 204 can be moved over the sample container in the sample drawer 210, and then pressed down and the needle 512 or 514 is inserted into the sample container.

[0163] In step 1808, the sample is transferred from the sample container to the processing tube in the sample preparation box by the first needle. In some embodiments, the needle 512 or 514 can draw the sample into the plastic body reservoir of the first pipette 202 or the second pipette 204, and the first pipette 202 or the second pipette 204 can be moved to the sample preparation box 500 to transfer the sample to the processing tube 510 in the sample preparation box 500.

[0164] To transfer the sample from the plastic body reservoir of the first pipette 202 or the second pipette 204 into the processing tube 510, the needle 512 or 514 (coupled to the first pipette 202 or the second pipette 204) can pierce the septum of the processing tube (e.g., the septum 704 of the processing tube 700). The first pipette 202 or the second pipette 204 can then dispense the sample into the processing tube through the needle 512 or 514.

[0165] In step 1810, the sample is concentrated and enriched for pathogens in the processing tube using the analyzer device, thereby producing an enriched sample in the processing tube. In some embodiments, the concentration and enrichment of the sample in step 1810 can be performed using components in the analyzer device 200 in a series of steps, including centrifugation, removal of fluids, addition of growth medium, and counting of pathogens in the enriched sample.

[0166] In step 1812, a plurality of aliquots of the enriched sample are dispensed into a plurality of reaction wells in an AST cartridge in the analyzer device. In some embodiments, the first pipette 202 or the second pipette 204 in the analyzer device 200 may dispense a plurality of aliquots of the enriched sample into the reaction wells 1214 in the AST cartridge 1200. In some embodiments, each aliquot may correspond to a corresponding reaction well 1214 in the AST cartridge 1200, and each reaction well 1214 may include a predetermined concentration of an antimicrobial agent for reacting with a corresponding aliquot of the enriched sample containing the pathogen.

[0167] In step 1814, the aliquots in the reaction wells of the AST box are incubated for a predetermined period of time to allow a reaction between the pathogens in the aliquots and the antimicrobial agent in each reaction well. In some embodiments, the first pipette 202 can move the AST box 1200 to the AST subsystem 250 or 1700, where the reaction wells 1214 can be placed in a heat block or heater for temperature-controlled incubation. In some embodiments, the predetermined period of time for the aliquots to be incubated in the reaction wells 1214 is about two hours or less.

[0168] In some embodiments, after incubating the aliquot in each reaction well 1214, the first pipette 202 can move the AST cartridge 1200 to a centrifuge (e.g., centrifuge 242 or 1412) in the analyzer device 200 to move the pathogen in each aliquot to the bottom wall of each reaction well 1214 to obtain an image of the AST cartridge 1200. In some embodiments, after incubating the aliquot in each reaction well 1214, the first pipette 202 can move the AST cartridge 1200 to the enrichment subsystem 1500, where a magnet (such as a magnet station) can be used to move the pathogen in each aliquot to the bottom wall of each reaction well 1214 to obtain an image of the AST cartridge 1200. In some embodiments, the pathogen can be attached to the magnetic beads in each aliquot.

[0169] In step 1816, an image of each reaction well in the AST box is acquired by using a microscope in the analyzer device. In some embodiments, the first pipette 202 can move the AST box 1200 to the AST subsystem 1700, where the AST box 1200 is imaged by the microscope 1702 using the scanning platform 1706. In some embodiments, each reaction well 1214 includes a bottom wall having an inner surface and an outer surface, wherein the reaction between the pathogen in the aliquot and the antimicrobial agent in each reaction well 1214 occurs above the inner surface of the bottom wall of each reaction well 1214 in the AST box 1200. In some embodiments, an image of the AST box 1200 is acquired at the outer surface of the bottom wall of each reaction well 1214 in the AST box 1200 by the microscope 1702. In some embodiments, the microscope 1702 is configured to acquire one or more images taken from below the outer surface of the AST box 1200. In some embodiments, the microscope 1702 can 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 box 1200.

[0170] In step 1818, the sensitivity of the pathogen in each reaction well to the antimicrobial agent is determined by analyzing the image. In some embodiments, the sensitivity of the pathogen in each reaction well 1214 to the antimicrobial agent can be determined by analyzing one or more images obtained from the microscope 1702 using a processor (e.g., processing device 116) coupled to the microscope 1702 in the analyzer device 200. In some embodiments, the processor can analyze one or more images by counting the number of fluorescent pathogens in each reaction well 1212 in the AST box 1200. In some embodiments, the processor can determine the sensitivity of the pathogen by determining whether one or more of the pathogens are sensitive, moderately sensitive, or resistant to the antimicrobial agent in each reaction well 1214 based on the staining of the cells in the aliquot by the first fluorescent dye.

[0171] In some embodiments, the processor can determine the sensitivity of the pathogen by determining whether one or more of the pathogens are sensitive, intermediately sensitive, or resistant to the antimicrobial agent in each reaction well 1214 based on the staining of the cells in the aliquot with the first fluorescent dye and / or the second fluorescent dye. In some embodiments, the processor can determine that a particular pathogen is resistant to a particular antimicrobial agent in reaction well 1214 by determining that a ratio of the number of dead cells to the number of live cells is below a predefined threshold for a predetermined concentration of the particular antimicrobial agent based on the staining of the cells in the aliquot with the first fluorescent dye and / or the second fluorescent dye. In some embodiments, the processor can determine that the ratio of the number of dead cells to the number of live cells is below a predefined threshold of about 20% or about 10%.

[0172] In some embodiments, a processor coupled to the microscope 1702 can be configured to detect highly resistant pathogens based on an accelerated reaction in one or more reaction wells 1214 caused by using a high dose of an antimicrobial in the reaction well 1214. In particular, a first reaction well 1214 in a plurality of reaction wells 1214 can contain an antimicrobial at a first concentration, wherein the first concentration is a high dose significantly higher than the clinical breakpoints of the antimicrobial and the pathogen. In some embodiments, the clinical breakpoint can represent the concentration or dose of an antimicrobial or antibiotic, which is used to define whether an infection of a specific pathogen is sensitive to successful treatment with the antimicrobial or antibiotic. In some embodiments, the use of an antimicrobial concentration higher than the clinical breakpoint can accelerate the reaction between the antimicrobial and the concentrated pathogen in the aliquot in the reaction well. In some embodiments, by using a higher antimicrobial concentration, the processor can be configured to determine the sensitivity of the pathogen by comparing the image of the first reaction well with the control in about one hour or less to determine the highly resistant pathogen.

[0173] In addition to determining pathogen sensitivity, the processor can be further configured to determine the minimum inhibitory concentration (MIC) of a specific antimicrobial agent for inhibiting the growth of a specific pathogen in one or more pathogens based on the image of the AST box 1200. In addition, the processor can be configured to determine whether a specific pathogen is sensitive, moderately sensitive or resistant to a specific antimicrobial agent based on the MIC value of the specific antimicrobial agent by parsing a rule set in a database (e.g., database 110) coupled to the processor communication. In some embodiments, one or more databases 110 can be configured to store pathogen taxonomy data and / or outcomes from a previous pathogen identification workflow (e.g., performed by the analyzer device 200). In some embodiments, the processor can retrieve pathogen taxonomy data and / or outcomes from one or more databases 110, and use the retrieved data to determine one or more rules that should be applied to call resistance / intermediate resistance / sensitive from MIC.

[0174] Fig.19 A flow chart of a method 1900 for manufacturing or assembling an AST cassette according to an embodiment of the present disclosure is shown. In some embodiments, the method 1900 may describe the manufacture and / or assembly of an AST cassette (such as AST cassette 232, 1200), as described above with reference to Figure 3B-Figure 1 7. It should be understood that the operations shown in method 1900 are not exhaustive, and other operations may also be performed before, after, or between any of the operations shown. In various embodiments of the present disclosure, the operations of method 1900 may be performed in different orders and / or variations.

[0175] Fig.19 The method 1900 begins at step 1902, wherein a cover comprising a plurality of openings is manufactured. In some embodiments, the cover 1202 of the AST box 1200 having openings 1206 can be manufactured using injection molding. In some embodiments, the cover 1202 can be made of polypropylene (PP), polycarbonate (PC), or another plastic material.

[0176] In step 1904, the diaphragm is overmolded into the cover so that the first side of the diaphragm extends across the plurality of openings in the cover. In some embodiments, the cover 1202 can be a substrate, and the diaphragm 1208 is molded directly on top of it to form a single solid piece. In some embodiments, the diaphragm 1208 can be made of a double layer of polytetrafluoroethylene (PTFE) and another material selected from silicone, rubber, and butyl rubber. In some embodiments, the diaphragm 1208 can be made of at least one of rubber, polytetrafluoroethylene (PTFE), thermoplastic elastomer (TPE), silicone, butyl rubber, or a combination thereof.

[0177] In step 1906, a base comprising a plurality of reaction holes may be produced. In some embodiments, base 1212 may be manufactured using injection molding. In some embodiments, base 1212 may be made of polystyrene (PS). In some embodiments, the production of base 1212 includes producing a plurality of reaction holes 1214 connected together as a single component, and in some embodiments, a plurality of reaction holes 1214 in base 1212 may be manufactured to have a conical shape. In some embodiments, the diameter of the bottom wall of each reaction hole 1214 may be less than about 2 mm.

[0178] In some embodiments, a predetermined concentration of antimicrobial agent can be added to each reaction well 1214 in liquid form during the manufacture of the base 1212 and before the base 1212 is attached to the diaphragm 1208. In some embodiments, forced air can be used to dry the antimicrobial agent in each reaction well 1214 to obtain a dry or freeze-dried form of the antimicrobial agent. In some embodiments, the steps of adding the antimicrobial agent and drying the antimicrobial agent in each reaction well 1214 can be completed within a predetermined time period to prevent degradation of the antimicrobial agent. In some embodiments, the predetermined time period for adding and drying the antimicrobial agent is about 10 minutes, 15 minutes, etc.

[0179] In step 1908, the base can be attached to the second side of the septum so that the second side of the septum extends across and seals the plurality of reaction wells in the base. In some embodiments, the first side of the septum 1208 is overmolded into the cap 1202, and the second side of the septum 1208 is attached to the base 1212 to provide a sealing mechanism on the reaction wells 1214 in the base 1212. In some embodiments, the base 1212 can be attached to the second side of the septum 1208 by using at least one of a snap-fit ​​joint or a mechanical fastener. In some embodiments, during the attachment of the base 1212 to the second side of the septum 1208, each opening 1206 in the cap 1202 can be aligned with a corresponding reaction well 1214 in the plurality of reaction wells 1214 in the base 1212. Example use cases

[0180] In some embodiments, the above-mentioned AST system can be used to determine the minimum inhibitory concentration of the antibiotic sensitivity test of the blood sample. In some embodiments, the blood sample is added to the reaction tube containing the lysing agent. In some embodiments, the amount of the sample can depend on the size of the reaction tube used. For example, if the volume of the reaction tube is 15mL, the sample volume in the tube will be less than 15mL, such as 10mL. In some embodiments, additional reagents (such as proteases) can be added to the reaction tube to improve the lysis effect. For example, 100 μL-2mL of proteases can be added. In order to crack and concentrate the sample, the sample can be incubated under a low-speed rotation motion, and then centrifuged. The supernatant of the sample can be discarded until the sample of the preferred amount is left to prepare the matrix for the enrichment step. In some embodiments, the volume is maintained in the range of 200 μL-2mL after concentration. In some embodiments, the volume of about 500 μL is maintained after concentration.

[0181] In some embodiments, enrichment is optionally carried out.Before this enrichment, culture medium broth can be added and vortexed.Enrichment can be carried out at the enrichment temperature with rotation stirring for a preferred time period.In some embodiments, the total enrichment volume can be determined by the volume of the culture medium added to the volume of the concentrated sample used.In some embodiments, the enrichment volume is about 5mL (e.g., 500 μL concentrated sample+4500 μL culture medium).Technicians will recognize that other amounts and ratios can be used without departing from the present invention.

[0182] In order to separate the target bacteria from the blood in the sample, in some embodiments, immunomagnetic separation can be used. For example, magnetic beads attached to biotinylated molecules associated with the target bacteria can be used. These coated magnetic beads can be added to the concentrated sample in the reaction tube and incubated. In some embodiments, non-specific magnetic beads can be used to capture any bacteria available in the sample.

[0183] In some embodiments, after capturing bacteria with magnetic beads, a reagent set can be used to digest, reduce and / or eliminate debris from the blood sample. As an example, a protease in the range of 2 μL-200 μL can be used for this purpose, and / or a DNase is used to digest any cell-free DNA.

[0184] After incubation, the collection of magnetic beads can be carried out by placing the reaction tube on or near the magnet. Once the beads are collected in the part closest to the magnet of the reaction tube, any supernatant can be sucked out and discarded. The remaining beads containing sample bacteria can be washed according to the cleaning process. Once washing is completed, the beads can be resuspended with culture medium. The desired amount of beaded samples (for example, 500 μL) can be extracted from the tube to a sample plate (such as a well plate), diluted as needed, and vortexed to reach the sample of the desired volume and concentration for incubation, and delivered to the multiple reaction wells in the plate. The sample plate can then be centrifuged to drive bacteria to the bottom of the hole for imaging. In some embodiments, the sample plate is centrifuged for 1 minute with a rotation of 3000G.

[0185] The samples on the sample plate are then incubated for the desired amount of time. For example, the samples can be incubated for 5 hours. The sample plate can then be centrifuged to drive the bacteria to the bottom of the wells for imaging. The incubated samples can then be imaged through the bottom of the sample plate to determine the growth of the sample and only the antimicrobial sensitivity. Experimental Example:

[0186] As discussed below, several experiments were performed to test various embodiments of the antibiotic susceptibility testing systems and methods described above. Example 1: Determination of Minimum Inhibitory Concentration from Blood Tube Samples

[0187] This example demonstrates how antibiotic susceptibility testing can be performed starting from a blood tube sample according to embodiments disclosed herein.

[0188] First, the beads coated with streptavidin are washed and fixed. The beads attached to biotinylated molecules (such as antibodies in this case) are incubated overnight with phosphate saline buffer (adjusted to pH 7.4) at room temperature with gentle rotation (25rpm). The typical binding capacity of each mg (100 μL) of magnetic beads used is about 20 μg of biotinylated antibodies. After coating, some washings are carried out with phosphate saline buffer containing 0.01% BSA (w / v) (pH 7.4 of adjustment). Finally, the beads are resuspended to the desired application concentration. The antibody used is biotin Klebsiella (Klebsiella) polyclonal antibody (PA1-73177, Invitrogen), and the magnetic beads used are Dynabeads MyOne Streptavidin T1 (65601, Thermofisher Scientific).

[0189] Blood samples were prepared from a single large blood pool from a donor collected in a 10 mL EDTA blood tube, which was spiked with an appropriate amount of bacteria (Klebsiella pneumoniae ATCC 13883) according to the target inoculum (approximately 100 CFU / 10 mL). After the pool was spiked, 10 mL was transferred to a lysing agent (700 μL isolating agent BC0507C, Oxoid) and immiscible fluorocarbon oil (20 μL Fluorinert TM FC-40 F9755, Sigma-Aldrich) in a previously labeled 15mL tube. For cracking and concentration, the sample was incubated for a short period of time (30 seconds) under a low speed (10rpm) rotary motion, and centrifuged for 5 minutes under a force of 12.000g with a fixed angle rotor. The supernatant of each sample was discarded until 500 μL for the preparation of matrix for the enrichment step. At this time, half of the samples were stopped, and these 500 μL were plated on agar plates as a checkpoint to understand how the concentration process was carried out and how many CFU to start the next stage.

[0190] For samples that continued the experiment, 4500mL of culture medium (Mueller Hinton broth) was added before the enrichment step and vortexed for 1 minute. This enrichment process was carried out for 30 minutes at 37°C and low speed (10rpm) rotation agitation. Once this stage is complete, reservoir dilutions are carried out and agar plates are plated to understand the bacterial population in each sample, to understand the replication time and bacterial quantity that can be used for the immunomagnetic separation process.

[0191] In order to immunomagnetically separate bacteria from blood, 100 μL antibody-coated beads were added to each sample tube. The incubation conditions used were to follow a gentle rotational motion (25rpm) for 30 minutes at 37°C. After incubation, in order to understand how many bacteria (combined with beads and uncombined), the reservoir dilution using 100 μL matrix was plated on LB agar plates. The collection of magnetic beads was carried out by placing the tube on a magnet for 15 minutes. After this time, aspirate and discard the supernatant, and wash with two washing steps to clean the sample. Each washing step involves adding 5mL washing buffer (phosphate saline buffer, pH 7.4), mixing gently for 1 minute at 37°C with a rotational motion (10rpm), collecting beads by placing the tube on a magnet for a short period of time (1 minute), and aspirate and discard the supernatant. Once washing is complete, the beads are resuspended and vortexed for 10-20 seconds with 500 μL culture medium (broth adjusted by Mueller Hinton cations). In order to determine the amount of bacteria captured by magnetic beads, 100 μL final matrix was used to prepare the dilution library, and on the LB agar plate, it was grown at 37°C for 16-18 hours. These samples were also observed under an inverted fluorescence microscope to understand the bacteria present in the sample and calculate the amount required for the AST test, which is the next and last step of this end-to-end assay. Each sample of 5 μL was transferred to a 384-well plate with a flat polystyrene film bottom, and incubated with fluorescent dye (SYBR green and propidium iodide) for 10 min in the dark at room temperature. The 384-well plates were centrifuged for 1 minute at 3000g in a swinging bucket motion, and observed in an inverted fluorescence microscope. For each sample, a composite image covering the bottom of each hole was constructed.

[0192] Based on this measurement, the sample was diluted (Mueller Hinton cation adjusted broth) to obtain approximately 4x10 4 The bacterial samples were incubated at 37°C with different ciprofloxacin concentrations at 2x10 4 CFU / mL final concentration was incubated for 5 hours. The 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.

[0193] Samples were imaged at incubation time 0 and after 5 hours of incubation (with and without antibiotics). 5uL 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 minutes at room temperature in the dark. The 384-well plate was centrifuged at 3000g for 1 minute in a swing bucket centrifuge and observed in an inverted fluorescence microscope. For each sample, a composite image was constructed. The images were analyzed with custom software to count bacteria, detect filamentous bacteria, count red bacteria, and record the brightness of the bacteria. These images are displayed in Fig.21A middle.

[0194] Data obtained from the images were used to construct a graph showing growth at 0.008 and 0.004 mg / L ciprofloxacin, but no growth at concentrations ≥ 0.015 mg / L ( Fig.21B ). These results, together with the bacterial counts and data analysis, indicate that the MIC for this strain is 0.015 mg / L, which is consistent with the MIC of 0.03 mg / L for this strain based on the broth microdilution method. This graph is presented in Fig.21B middle. Example 2: Determination of Minimum Inhibitory Concentration for Samples from Blood Culture Bottles

[0195] This embodiment demonstrates how to perform antibiotic susceptibility testing starting from a blood culture bottle according to embodiments disclosed herein.

[0196] First, the beads coated with streptavidin are washed and fixed. Washing buffer and fixation process can be different according to application. The beads attached to biotinylated molecules (such as antibodies in this case) are incubated overnight with phosphate saline buffer (adjusted to pH 7.4) at room temperature with gentle rotation (25rpm). The typical binding capacity of each mg (100 μL) of magnetic beads used is about 20 μg biotinylated antibodies. After coating, some washings are carried out with phosphate saline buffer containing 0.01% BSA (w / v) (pH 7.4 of adjustment), and then the beads are finally resuspended to the desired application concentration. The antibody used is a biotin Klebsiella polyclonal antibody (PA1-73177, Invitrogen), and the magnetic beads used are Dynabeads MyOne Streptavidin T1 (65601, Thermofisher Scientific).

[0197] Blood samples were prepared from a single large pool of previously mixed blood that was spiked with the appropriate amount of bacteria (Klebsiella pneumoniae ATCC13883) according to the target inoculum (approximately 100 CFU). After the pool was spiked, 10 mL was transferred to a previously labeled blood culture system (442023, BD BACTECTM Plus Aerobic / F) and mixed with a stirring platform (5 minutes at 150 rpm). Once the time has passed, wait a reasonable time (30 seconds-1 minute) for the resin to settle. Then extract a 5 mL sample.

[0198] The immunomagnetic separation of bacteria was performed using a 15mL tube containing 5mL of matrix extracted from a blood culture bottle and 100 μL of beads coated with anti-Klebsiella pneumoniae antibodies. The incubation conditions used were at 37°C and with gentle rotation (25rpm) for 30 minutes. After incubation, 100 μL of matrix was used to dilute the reservoir and to plate to understand how many bacteria (bacteria combined with beads and uncombined) were contained in each sample. The collection of magnetic beads was performed by placing the tube on a magnet for 7.5 minutes. After this time, the supernatant was aspirated and discarded, and three washing steps were performed to wash with clean samples. Each washing step involves adding 5mL of culture medium (broth adjusted by Mueller Hinton cations), vortexed for 1 minute, gently mixed for 2 minutes at 37°C with slow rotation (10rpm), collected beads by placing the tube on a magnet for a short period of time (2 minutes), and aspirated and discarded the supernatant. After washing, the beads are resuspended with 500 μL culture medium (broth adjusted by Mueller Hinton cations) and vortexed for 1 minute. In order to determine the amount of bacteria captured by the antibody-coated magnetic beads, 100 μL final matrix is ​​used to prepare the dilution library, and the plating is carried out on LB agar plates to grow for 16-18 hours at 37 ° C. Samples are also observed under an inverted fluorescence microscope to determine the number of bacteria present in the sample and to calculate the required adjustment for AST test. Each sample of 5 μL is 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. 384-well plates are centrifuged in a swing bucket centrifuge for 1 minute at 3000g, and observed in an inverted fluorescence microscope. For each sample, a synthetic image covering the bottom of each hole is constructed. The image is analyzed with custom software to determine the number of bacteria.

[0199] Based on this measurement, the sample was diluted (Mueller Hinton cation adjusted broth) to obtain approximately 4x10 4 The number of bacteria was expressed as CFU / mL. Bacterial samples were incubated at 37°C in a final volume of 10 μL at 2 × 10 4 CFU / mL and incubated for 5 hours. The gentamicin concentrations tested were 2, 1, 0.5, 0.25, 0.125 and 0.06 mg / L. Three replicates were performed for each antibiotic concentration.

[0200] Samples were analyzed after incubation time 0 and 5 h of incubation (with and without antibiotics). 5 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 minutes at room temperature in the dark. The 384-well plate was centrifuged at 3000 g for 1 minute in a rocking bucket motion and observed in an inverted fluorescence microscope. For each sample, a composite image was constructed. The images were analyzed with custom software to count bacteria, detect filamentous bacteria, count red bacteria, and record the brightness of the bacteria. These images are displayed in Fig.22A middle.

[0201] Data obtained from the images were used to construct a graph showing growth at 0.125 and 0.06 mg / L gentamicin, but no growth at concentrations ≥ 0.25 ( Fig. 22B ). These results, together with the bacterial counts and data analysis, indicate that the MIC for this strain is 0.25 mg / L, and based on the broth microdilution method, the MIC for this strain is 0.5-0.25 mg / L. This chart is presented in Fig. 22B middle. Example 3: Determination of the minimum inhibitory concentration of gentamicin for Gram-negative bacteria.

[0202] The embodiments disclosed herein were used to determine the minimum inhibitory concentration of a strain of the Gram-negative bacterium Klebsiella pneumoniae (ATCC13883) that is sensitive to gentamicin (MIC of 0.25-0.5 mg / L according to the broth microdilution method). The bacteria were grown in liquid culture until the exponential phase appeared in the culture medium. The culture was diluted in culture medium (MuellerHinton cation-adjusted broth) and incubated at 37°C with different concentrations of gentamicin at 2 x 10 4 CFU / mL and incubated for 5 hours. The gentamicin concentrations tested were 4, 2, 1, 0.5, 0.25 and 0.125 mg / L. Three replicates were performed for each antibiotic concentration.

[0203] Samples were analyzed at incubation time 0 and after 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 (NaCl 0.9%) and transferred to a 384-well plate with a flat polystyrene membrane bottom. The 384-well plate was centrifuged at 3000g for 1 minute in a rocking bucket motion and observed in an inverted fluorescence microscope. For each sample, a composite image covering the bottom of each well was constructed. The images were analyzed with custom software to count bacteria, detect filamentous bacteria, count red bacteria, and record the brightness of the bacteria. These images are shown in Fig.23A middle.

[0204] Data obtained from the images were used to construct a graph showing that bacteria grew at 0.125 mg / L gentamicin but did not grow at concentrations ≥ 0.25 mg / L ( Fig. 23B ). These results, together with the bacterial counts and data analysis, indicate that the MIC for this strain is 0.25 mg / L, which corresponds to the MIC obtained by the broth microdilution method. The results show that the MIC of these Gram-negative strains to gentamicin can be determined by incubating for 5 hours at different antibiotic concentrations and using bacterial growth and bacterial characteristics. This chart is presented in Fig. 23B middle. Example 4: Determination of the minimum inhibitory concentration of meropenem against Gram-negative bacteria.

[0205] The minimum inhibitory concentration of a strain of the Gram-negative bacterium Klebsiella pneumoniae (IHMA1977064) with a MIC of 8 mg / L for meropenem according to the broth microdilution method was determined using the embodiments disclosed herein. The bacteria were grown in liquid culture until the exponential phase appeared in the culture medium. The culture was diluted in culture medium (Mueller Hinton cation-adjusted broth) and incubated at 37°C with different concentrations of meropenem at 2 x 10 4 CFU / mL and incubated for 5 hours. The meropenem concentrations tested were 32, 16, 8, 4, 2 and 1 mg / L gentamicin. Three replicates were performed for each antibiotic concentration.

[0206] Samples were analyzed after incubation time 0 and 5 hours of incubation (with and without antibiotics). 5uL 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 minutes at room temperature in the dark. The 384-well plate was centrifuged at 3000g for 1 minute in a swinging bucket motion and observed in an inverted fluorescence microscope. For each sample, a composite image covering the bottom of each well was constructed. The image was analyzed with custom software to count bacteria, detect filamentous bacteria, count red bacteria, and record the brightness of the bacteria. These images are displayed in Fig.24A middle.

[0207] Data obtained from the images were used to construct a graph showing that bacteria grew at 2 and 1 mg / L meropenem, but not at concentrations ≥ 4 mg / L ( Fig. 24B ). These results, together with the bacterial counts and data analysis, indicate a MIC of 4 mg / L, which is consistent with the MIC of 8 mg / L for this strain based on the broth microdilution method. This graph is presented in Fig. 24B middle.

[0208] The results showed that the MIC of these Gram-negative strains to meropenem could be determined by incubating for 5 hours at different antibiotic concentrations and using bacterial growth and bacterial characterization. Example 5: Determination of the minimum inhibitory concentration of erythromycin for Gram-positive bacteria

[0209] According to the embodiments described herein, the minimum inhibitory concentration of two strains of the Gram-positive bacterium Staphylococcus aureus (Staphylococcus aureus), one sensitive to erythromycin (ATCC29213) and one resistant to erythromycin (ATCC43300), was determined. The MIC of these strains was obtained by the microbroth dilution method, and the MIC of Staphylococcus aureus (S. aureus) sensitive to erythromycin was 0.25-0.5 mg / L, and the MIC of the resistant strain was ≥512 mg / L. The bacterial strains were grown in liquid culture until the exponential phase was reached in rich medium. The bacterial culture was diluted in a culture medium (Mueller Hinton cation-adjusted broth) and incubated at 37°C with 2x 10 in a final volume of 10 μL with different concentrations of gentamicin. 4 CFU / mL and incubated for 5 hours. Erythromycin-sensitive S. aureus strains were tested at 1, 0.5, 0.25, 0.125, 0.06 and 0.03 mg / L, while 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.

[0210] Samples were analyzed after incubation time 0 and 5 hours of incubation (with and without antibiotics). Samples were incubated in the dark for 10 minutes at room temperature with fluorescent dyes (SYBR green and propidium iodide). After dye incubation, 1 μL of sample was diluted with 4 μL buffer (NaCl 0.9%) and transferred to a 384-well plate with a flat polystyrene film bottom. The 384-well plate was centrifuged for 1 minute at 3000 g in a swinging bucket motion and observed in an inverted fluorescence microscope. For each sample, a composite image covering the bottom of each hole was constructed. Image was analyzed with custom software to count bacteria, detect filamentous bacteria, count red bacteria, and record the brightness of bacteria.

[0211] The data obtained from the images were used to plot the susceptibility and resistance of Staphylococcus aureus to erythromycin ( Fig.25A and Fig.25B ). Staphylococcus aureus, which is sensitive to erythromycin, showed growth at 0.125 mg / L erythromycin but no growth at concentrations ≥ 0.25 mg / L ( Fig.25A). These results, together with the bacterial counts and data analysis, indicate that the MIC for this strain is 0.25 mg / L, which corresponds to the MIC obtained by the broth microdilution method. Data analysis of erythromycin-resistant Staphylococcus aureus strains ( Fig.25B ) showed growth at 4 mg / L and lower concentrations of erythromycin, indicating that the MIC of this strain is ≥4 mg / L. The MIC obtained by the broth microdilution method is ≥512 mg / L, indicating that with both methods, this strain is resistant to erythromycin (EUCAST and CLSI breakpoints indicate that Staphylococcus aureus strains with MIC>2 are resistant to erythromycin). The results show that the MIC of these Gram-positive strains to erythromycin can be determined by incubating for 5 hours at different antibiotic concentrations and using bacterial growth and bacterial characteristics. Example 6: Rapid determination of bacteria resistant to ampicillin

[0212] In order to quickly detect highly resistant bacteria to a specific antimicrobial, bacteria were grown for a short period of time (1 hour) in the presence of a high dose of the antimicrobial. Two strains of E. coli (one sensitive to ampicillin (ATCC25922) and the other resistant to ampicillin (ATCC700891)) were grown in culture until exponential phase. Bacterial cultures were diluted in culture medium (Mueller Hinton cation-adjusted broth) and incubated at 37°C at 2x10 in a final volume of 10 μL in the presence of ampicillin at a final concentration of 128 mg / L, 64 mg / L, and 32 mg / L. 4 CFU / mL final concentration was incubated for 1 h. Three replicates were performed for each antibiotic concentration.

[0213] Samples were visualized at time 0 before any incubation of representative bacteria and after 1 hour of incubation. 5 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 minutes in the dark at room temperature. The 384-well plate was centrifuged at 3000g for 1 minute in a rocking bucket motion and observed in an inverted fluorescence microscope. For each sample, a composite image covering the bottom of each well was constructed. The image was analyzed with custom software to count bacteria, detect filamentous bacteria, count red bacteria, and record the brightness of the bacteria. These images are displayed in Fig.26A middle.

[0214] When comparing the profiles of the two E. coli strains at different ampicillin concentrations, the ampicillin-sensitive E. coli strain with an MIC of 4 mg / L according to the broth microdilution method did not grow at any of the ampicillin concentrations tested ( Fig.26B), at the three concentrations tested, the bacteria were in filamentous form, and red bacteria were also present at 128 mg / L of ampicillin, indicating that the bacteria were dead. However, according to the broth microdilution method, the ampicillin-resistant E. coli strains with MIC>128 mg / L showed growth after 1 hour at all three tested ampicillin concentrations, and there were no filamentous bacteria or red bacteria ( Fig.26B ).

[0215] The results showed that bacteria resistant to ampicillin could be detected by incubating at high antibiotic concentrations for only 1 hour and using bacterial growth and bacterial characterization. Example 7: Cleaning up blood samples with specific beads

[0216] Different time conditions are evaluated to establish the best duration of magnetic beads to capture bacteria. First, based on application, the beads coated with streptavidin are washed with washing buffer. Once washed, the beads are fixed. The beads are attached to biotinylated molecules (such as antibodies), as in this case, with phosphate saline buffer (adjusted to pH 7.4) at room temperature accompanied by gentle rotation (25rpm) for overnight incubation. The typical binding capacity of the tested magnetic beads of every mg (100 μL) is about 20 μg of biotinylated antibodies. After completing the coating, some washings are carried out with containing 0,01% BSA (phosphate saline buffer of w / v) (adjusted to pH 7,4), and finally the beads are resuspended to the desired concentration for application. The antibody used is a biotin Klebsiella polyclonal antibody (PA1-73177, Invitrogen), and the magnetic beads used are Dynabeads MyOne Streptavidin T1 (65601, Thermofisher Scientific).

[0217] Different capture times were evaluated with blood samples. Blood samples were prepared from a single large pre-oscillation blood pool, which was spiked with an appropriate amount of bacteria (Klebsiella pneumoniae ATCC13883) according to the target inoculum. After the pool was spiked, 10 mL was transferred to a previously labeled blood culture system (442023, BD BACTEC TM Plus Aerobic / F) and mixed with a stirring platform (5 minutes at 180 rpm). Once the time has passed, wait a reasonable time (30 seconds-1 minute) to allow the resin to settle and extract 5 mL of the sample. No more than 10 mL is extracted from each blood culture system.

[0218] 5mL blood is transferred to 15mL tube from blood culture system, and the antibody-coated beads (100 μL) of required amount are added.At 37 ℃, follow gently rotating motion (25rpm) to hatch for 30 minutes.After hatching, 100 μL matrix is ​​used to carry out reservoir dilution, and its bed plate.By placing the tube on magnet for the time (15-7.5 minutes) of continuous test, collect beads, and then supernatant is sucked out and discarded.At this moment, add 5mL washing buffer to start the washing of three corresponding batches.Each washing step comprises vortexing 1 minute, and the sample is hatched for 2 minutes at 37 ℃ with gently rotating motion (25rpm), by placing the tube on magnet for a short period of time (5-2,5 minutes) to collect beads, and suck out and discard supernatant.After washing, beads are resuspended with 500 μL culture medium (broth adjusted by Mueller Hinton cations) and vortexed for 1 minute.The next step comprises using 100 μL matrix to carry out reservoir dilution and its bed plate. In parallel, these samples were also visualized under a microscope to check whether the two types of readings (plates compared to microscopes) were consistent. In order to observe the samples under an inverted fluorescence microscope, 5 μL of each sample was transferred to a 384-well plate containing a fluorescent dye (SYBR Green + propidium iodide). Subsequently, the plate was incubated at room temperature for 10 minutes and a centrifugation process (3000 g force for 1 minute) was performed and then visualized. Table 1 below shows that the results were very little dependent on the capture time: Table 1 Example 8: Blood sample cleanup process using non-specific beads

[0219] A series of reagents have been evaluated to improve sample cleanup of cell debris from blood cell lysates. Blood samples were prepared from a single large shaken pre-blood (5 minutes at 150 rpm). After shaking, 10 mL was transferred to a previously labeled blood culture system (442023, BD BACTEC TM Plus Aerobic / F) and mix with a stirring platform (5 minutes at 180 rpm). Once the time has passed, wait a reasonable time (30 seconds-1 minute) for the resin to settle and extract 10 mL of the sample. No more than 10 mL is extracted from each blood culture system.

[0220] A 10 mL blood sample extracted from a blood culture system was transferred to a 15 mL tube for lysis with a variable volume of lysis agent (700 μL of 2.8 g / 100 mL saponin S4521, Sigma-Aldrich). The tube used for the lysis and concentration steps also contained an immiscible fluorocarbon oil (20 μL Fluorinert TMFC-40F9755, Sigma-Aldrich). In addition to the lysing agent, other reagents such as proteases are also added to increase their effects (250 μL of proteases from Aspergillus oryzae P6110, Sigma-Aldrich). In order to perform the lysis and concentration steps, it is necessary to incubate the sample for a short period of time (30 seconds) under a low rpm (10 rpm) rotational motion, and then centrifuge for 5 minutes at 12000 g to discard the supernatant until 500 μL, to prepare a matrix for IMS containing 500 μL TTGB 2X buffer. Once the initial matrix is ​​obtained, other complementary reagents such as DNA enzymes (different concentrations and times) are added to improve the elimination of cell debris. Before adding magnetic beads, the sample is spiked with an appropriate amount of bacteria (Escherichia coli ATCC25922) according to the target inoculum. After the matrix is ​​spiked, a short vortex is performed. At this time, magnetic beads are added, but not before gently mixing.

[0221] The magnetic beads used are combined with peps6 peptides, which are synthetic forms of ApoH proteins, which are blood-derived human proteins (MP10031, ApoH Technologies) that are considered to be potential infectious products. The conditions for immunomagnetic separation with these beads are 30 minutes at 37°C with gentle rotation (25rpm) and 20 degrees of inclination. Once binding occurs, 4mL 1X TTGB buffer is added, 100 μL samples are used for reservoir dilution, and agar plates are used to determine the number of bacteria before the magnet is maintained. The desired time for correct magnetic separation is established in about 15 minutes. Subsequently, the supernatant is eliminated and 4 washing batches are performed. Each washing step includes adding 5mL washing buffer (broth adjusted by Mueller Hinton cations), vortexing for 1 minute, and the sample is incubated for 2 minutes at 37°C with gentle rotation (25rpm), and the beads are collected by placing the tube on the magnet for a short period of time (5 minutes), and the supernatant is sucked out and discarded.

[0222] After washing, the beads are resuspended with 500 μL culture medium (broth adjusted by Mueller Hinton cations), vortexed for 1 minute, and each sample of 250 μL is transferred to two 1.5mL tubes. At this point, a second treatment with reagent is performed to further clean the sample and to disaggregate the possible aggregates detected in the previous assay. Specifically, 10 μL proteases are added and left to stand for 30 seconds. In order to avoid the effect of proteases on bacteria, proteases are eliminated by centrifugation at 1000g force for 1 minute with a fixed angle rotor. Finally, the supernatant is discarded, and the beads are resuspended with 500 μL (broth adjusted by Mueller Hinton cations) and vortexed for 1 minute. Agar plates using 100 μL matrix and all samples are used for reservoir dilution, and samples are also observed under a microscope. Fig. 27 Images of samples with and without protease are shown.

[0223] 5 μL of each sample was transferred to a 384-well plate containing a certain amount of dye (SYBR Green + PI), incubated at room temperature for 10 minutes, and subjected to centrifugation (3000 g force for 1 minute) before visualization.

[0224] Table 2 below shows that a significantly larger fraction of bacteria was recovered at the end of the cleaning process and that the count numbers were higher when protease was used after washing, most likely due to the disaggregation of bacterial aggregates. Table 2 Exemplary Computer System:

[0225] Fig. 20 is a block diagram of exemplary computer system 2000 components. For example, one or more computer systems 2000 can be used to implement any of the embodiments discussed herein and combinations and sub-combinations thereof. In some embodiments, one or more computer systems 2000 can be used for image acquisition, image analysis, and data processing, such as for a microscope or processing device 116 in an AST subsystem 1700, as described herein. In some embodiments, one or more computer systems 2000 can also be used in a controller 109 to program and operate the movement of various components in an analyzer device 200. The computer system 2000 may include one or more processors (also referred to as central processing units or CPUs), such as a processor 2004. The processor 2004 can be connected to a communication infrastructure or bus 2006.

[0226] The computer system 2000 may also include one or more user input / output interfaces 2002 , such as a monitor, keyboard, pointing device, etc., which may communicate with the communication infrastructure 2006 via one or more user input / output devices 2003 .

[0227] One or more processors 2004 may be a graphics processing unit (GPU). In an embodiment, a GPU may be a processor that is a dedicated electronic circuit designed to process mathematically intensive applications. A GPU may have a parallel structure that efficiently processes large blocks of data in parallel, such as mathematically intensive data commonly found in computer graphics applications, images, videos, etc.

[0228] 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 sensitivity testing, and generate recommendations for treatment of the patient accordingly.

[0229] The computer system 2000 may also include one or more secondary storage devices or memories 2010. The secondary storage 2010 may include, for example, a hard drive 2012 and / or a removable storage drive 2014.

[0230] The removable storage drive 2014 can interact with the removable storage unit 2018. The removable storage unit 2018 can include a computer-usable or readable storage device having computer software (control logic) and / or data stored thereon. The removable storage unit 2018 can be a program cartridge and cartridge interface (such as found in a video game device), a removable storage chip (such as an EPROM or PROM) and associated sockets, a memory stick and USB port, a memory card and associated memory card slot, and / or any other removable storage unit and associated interface. The removable storage drive 2014 can read from and / or write to the removable storage unit 2018.

[0231] Secondary storage 2010 may include other means, devices, components, tools, or other methods for allowing access to computer programs and / or other instructions and / or data by computer system 2000. Such means, devices, components, tools, or other methods may include, for example, a removable storage unit 2022 and an interface 2020. Examples of removable storage unit 2022 and interface 2020 may be a program cartridge and cartridge interface (such as found in video game devices), a removable storage 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 any other removable storage unit and associated interface.

[0232] The computer system 2000 may further include a communication 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 numeral 2028). For example, the communication interface 2024 may allow the computer system 2000 to communicate with an external or remote device 2028 via a communication path 2026, which may be wired and / or wireless (or a combination thereof) and may include any combination of a LAN, a WAN, the Internet, etc. Control logic and / or data may be transmitted to and from the computer system 2000 via the communication path 2026.

[0233] The computer system 2000 may also be any of the following: a personal digital assistant (PDA), a desktop workstation, a laptop or notebook computer, a netbook, a tablet computer, a smart phone, a smart watch or other wearable device, an appliance, part of the Internet of Things, and / or an embedded system (to name a few non-limiting examples) or any combination thereof.

[0234] Computer system 2000 can be a client or server, accessing or hosting any application and / or data through any delivery paradigm, including but not limited to remote or distributed cloud computing solutions; local or on-premises software ("on-premises" cloud-based solutions); "as a service" models (e.g., content as a service (CaaS), digital content as a service (DCaaS), software as a service (SaaS), hosted 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 services or delivery paradigms.

[0235] Any applicable data structures, file formats, and schemas in computer system 2000 may be derived from standards, including but not limited to JavaScript Object Notation (JSON), Extensible Markup Language (XML), another markup language (YAML), Extensible Hypertext Markup Language (XHTML), Wireless Markup Language (WML), message packages, XML User Interface Language (XUL), or any other functionally similar representation (alone or in combination). Alternatively, proprietary data structures, formats, or schemas may be used alone or in combination with known or open standards.

[0236] In some embodiments, a tangible non-transitory device or article of manufacture including a tangible non-transitory computer-usable or readable medium having control logic (software) stored thereon may also be referred to herein as a computer program product or program storage device. This includes, but is not limited to, computer system 2000, main memory 2008, secondary memory 2010, and removable storage units 2018 and 2022, and tangible articles of manufacture embodying any combination of the foregoing. When executed by one or more data processing devices (such as computer system 2000), such control logic may cause such data processing devices to operate as described herein.

[0237] Based on the teachings contained in this disclosure, it will be apparent to one or more persons skilled in the relevant art how to use the Fig. 20 It will be clear that data processing devices, computer systems, and / or computer architectures other than those shown can be used to make and use the embodiments of the present disclosure. In particular, the embodiments can be operated using software, hardware, and / or operating system implementations other than those described herein.

[0238] It should be understood that the detailed description section (rather than the summary and abstract sections) is intended to be used to interpret the claims. The summary and abstract sections may set forth one or more but not all exemplary embodiments of the present disclosure as contemplated by one or more inventors, and therefore are not intended to limit the present disclosure and the appended claims in any way.

[0239] Embodiments of the present disclosure have been described above with the aid of functional building blocks, which demonstrate the implementation of specified functions and their relationships. For ease of description, the boundaries of these functional building blocks are arbitrarily defined herein. As long as the specified functions and their relationships are appropriately performed, alternative boundaries can be defined.

[0240] The above description of specific embodiments will fully reveal the general nature of the present disclosure, and others can easily modify and / or adjust such specific embodiments for various applications without undue experimentation by applying knowledge within the technical scope of the art without departing from the general concept of the present disclosure. Therefore, based on the teachings and guidance given herein, such adjustments and modifications are intended to be included in the meaning and range of equivalents of the disclosed embodiments. It should be understood that the words or terms in this article are for the purpose of description rather than limitation, and therefore the terms or words of this specification will be interpreted by those skilled in the art based on the teachings and guidance.

[0241] 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. A method, comprising: receiving, by an analyzer device, a sample preparation cartridge and a sample container containing a sample comprising a pathogen; installing a first needle from the sample preparation cartridge in a pipette system in the analyzer device; inserting the first needle into the sample container using the pipette system; transferring at least a portion of the sample from the sample container to a processing tube in the sample preparation cartridge via the first needle; using the analyzer device to concentrate and enrich the transferred sample in the processing tube for pathogens, thereby producing an enriched sample in the processing tube; distributing a plurality of aliquots of the enriched sample into a plurality of reaction wells in an antimicrobial susceptibility test (AST) cartridge in the analyzer device, wherein each aliquot corresponds to a respective reaction well, and wherein each reaction well contains a predetermined concentration of an antimicrobial agent; 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 the aliquots and the antimicrobial agent in each reaction well; Acquire an image of each reaction well in the AST box by using a microscope in the analyzer device; as well as The images are analyzed by a processor coupled to a microscope in the analyzer device to determine the susceptibility of the pathogen to the antimicrobial agent in each reaction well.

2. The method of claim 1, wherein the sample container is a blood culture bottle in which the sample is incubated for an amount of time to allow the pathogen to grow but not reach a growth plateau. The method according to claim 1 , wherein the sample container is a blood sample tube.

4. The method according to claim 1, further comprising: identifying the number of pathogens in the transferred sample by using fluorescent dyes and a microscope in the analyzer device for marking and counting the pathogens; as well as In response to the identification, the transferred sample is enriched or diluted to obtain a predetermined amount of the pathogen in the enriched sample.

5. The method of claim 1, wherein concentrating pathogens from the transferred sample using the analyzer device comprises: moving the processing tube to a centrifuge in the analyzer device; centrifuging the processing tube in the centrifuge to concentrate pathogens in the transferred sample; as well as Fluid is removed from the process tube using the pipette system, leaving concentrated pathogens in the process tube.

6. The method of claim 5, wherein the sample is a blood sample, and the method further comprises, before centrifuging the processing tube: adding one or more lysis reagents to the process tube; and The one or more lysing reagents are mixed with the blood sample in the processing tube to lyse blood cells in the blood sample.

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

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

9. The method according to claim 5, further comprising: After an additional predetermined period of time, the concentrated pathogens are cleaned by centrifuging the process tube and removing the supernatant from the process tube to obtain the enriched sample.

10. The method of claim 5, wherein the processing tube further comprises magnetic beads configured to attach to concentrated pathogens in the processing tube, the method further comprising: retaining the concentrated pathogens attached to the magnetic beads in the processing tube by using a magnet station in the analyzer device; as well as Extraneous liquid is removed from the processing tube, thereby producing an enriched sample having the concentrated pathogens. The method according to claim 10 , wherein the magnetic beads are coated with a non-specific ligand.

12. The method of claim 10, wherein the magnetic beads are coated with a specific ligand that is specific for a particular pathogen of the concentrated pathogens in the processing tube.

13. The method of claim 10, further comprising adding treatment with protease and / or DNase after attaching the concentrated pathogens to the magnetic beads but before retaining the concentrated pathogens attached to the magnetic beads with the magnet.

14. The method according to claim 5, further comprising: One or more washing materials are added to the concentrated pathogens in the processing tube to clean and remove any blood components or debris from the concentrated pathogens, leaving an enriched sample in the processing tube.

15. The method of claim 14, wherein the one or more washing materials comprise a combination of one or more buffers, detergents, surfactants and proteases.

16. The method according to claim 1, further comprising: The antimicrobial agent is added in liquid form to each reaction well in the AST cartridge, and then aliquots of the enriched sample are dispensed into the reaction wells.

17. The method of claim 1, wherein the antimicrobial agent in each reaction well of the AST cartridge is in a dry form or a freeze-dried form prior to dispensing the plurality of aliquots of the enriched sample into the reaction wells.

18. A method according to claim 1, wherein dispensing the multiple aliquots to the multiple reaction wells includes using jet dispensing to perform non-contact dispensing of the aliquots through a second needle in the sample preparation box, wherein the second needle penetrates the corresponding diaphragms of the reaction wells and the second needle does not contact the bottom wall of each reaction well. The method of claim 18 , wherein the second needle is identical to the first needle.

20. The method of claim 18, wherein the volume of each aliquot dispensed through the second needle is in the range of about 0.5 microliters to about 10 microliters.

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

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

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

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

25. The method of claim 1, wherein the predetermined period of time for incubating the aliquot is about two hours or less.

26. The method of claim 1, further comprising: After incubating the aliquots in each reaction well, the AST cartridge is moved to a centrifuge in the analyzer device to move the pathogens in each aliquot to the bottom wall of each reaction well to acquire an image of the AST cartridge.

27. The method of claim 1, further comprising: After incubating the aliquots in each reaction well, the pathogens in each aliquot attached to the magnetic beads in each aliquot are moved to the bottom wall of each reaction well using a magnet in the analyzer device to acquire an image of the AST cartridge.

28. The method of claim 1, further comprising: Prior to acquiring the image, a first fluorescent dye is added to each reaction well to stain the pathogen in the aliquot of the reaction well.

29. The method of claim 28, wherein adding the first fluorescent dye to each reaction well comprises performing non-contact dispensing of the first fluorescent dye through a second needle in the sample preparation box using jet dispensing, the second needle penetrating the corresponding diaphragm of the reaction well and the second needle not contacting the bottom wall of each reaction well.

30. The method of claim 29, wherein the volume of the first fluorescent dye dispensed into each reaction well through the second needle is in the range of about 0.5 microliters to 10 microliters.

31. The method of claim 30, wherein the second needle is identical to the first needle.

32. The method of claim 28, wherein acquiring the image comprises acquiring one or more fluorescent images to detect a first fluorescent dye in each reaction well of the AST cartridge.

33. The method of claim 32, wherein the analysis of the image comprises counting the number of fluorescent pathogens in each reaction well in the AST box.

34. The method of claim 28, further comprising: Prior to acquiring the image, a second fluorescent dye was added to each reaction well.

35. The method of claim 34, wherein the first fluorescent dye comprises a DNA binding dye that labels live cells in an aliquot of the reaction well, and the second fluorescent dye comprises a fluorescent intercalator that labels dead cells or cannot permeate intact cell membranes in an aliquot of the reaction well.

36. A method according to claim 35, wherein determining the sensitivity of the pathogen comprises determining whether one or more of the pathogens are sensitive, intermediately sensitive or resistant to the antimicrobial agent in each reaction well based on staining of cells in the aliquot by the first fluorescent dye and / or the second fluorescent dye.

37. The method of claim 36, wherein determining that a particular pathogen is resistant to a particular antimicrobial agent in a reaction well comprises: Based on staining of cells in the aliquot by the first fluorescent dye and / or the second fluorescent dye, it is determined that the ratio of the number of dead cells to the number of live cells is below a predefined threshold for a predetermined concentration of a particular antimicrobial agent.

38. The method of claim 1, wherein determining the sensitivity of the pathogen comprises determining whether one or more of the pathogens is sensitive, intermediately sensitive, or resistant to the antimicrobial agent in each reaction well based on staining of cells in the aliquot by a first fluorescent dye.

39. The method of claim 1, wherein a first reaction well of the plurality of reaction wells comprises a first concentration of the antimicrobial agent, wherein the first concentration is a high dose that is above a clinical breakpoint for the antimicrobial agent and the pathogen.

40. The method of claim 39, wherein determining the sensitivity of the pathogen comprises comparing images of the first reaction well to a control within about one hour to determine highly resistant pathogens.

41. The method of claim 1, further comprising: A minimum inhibitory concentration (MIC) of a specific antimicrobial agent for inhibiting growth of a specific pathogen among the one or more pathogens is determined by the processor based on the image of the AST cartridge.

42. The method of claim 41, further comprising: The processor determines, by parsing a rule set in a database communicatively coupled to the processor, whether the specific pathogen is sensitive, moderately sensitive, or resistant to the specific antimicrobial agent based on the MIC value of the specific antimicrobial agent.

43. An antimicrobial susceptibility test (AST) kit, comprising: a base comprising a plurality of reaction wells, each reaction well comprising a bottom wall, wherein the bottom wall is optically transparent; a diaphragm disposed on the base, the diaphragm sealing each of the plurality of reaction wells, and a cover disposed on the diaphragm, Each reaction well of the plurality of reaction wells contains an antimicrobial agent at a predetermined concentration for reacting with a corresponding aliquot of the enriched sample comprising a pathogen, and wherein the antimicrobial agent is disposed within each reaction well.

44. The AST box of claim 43, wherein the antimicrobial agent in each reaction well is in liquid form.

45. The AST kit of claim 43, wherein the antimicrobial agent in each reaction well is in a dried form or a freeze-dried form.

46. ​​An AST box according to claim 43, wherein the bottom wall of each reaction well in the plurality of reaction wells is configured for optical interrogation.

47. The AST box of claim 43, wherein the bottom wall of each reaction well in the plurality of reaction wells is configured for fluorescence microscopy.

48. An AST box according to claim 43, wherein the diameter of the bottom wall of each reaction hole is less than about 2 mm.

49. The AST box of claim 43, wherein each reaction hole comprises a conical shape.

50. The AST box according to claim 43, wherein each reaction well is configured to receive an aliquot of the enriched sample containing pathogens, by non-contact dispensing of the aliquot by penetrating the diaphragm of the reaction well with a needle but without contacting the bottom surface of each reaction well.

51. An AST box according to claim 43, wherein the cover includes a plurality of openings, each opening being aligned with a corresponding reaction well among a plurality of reaction wells in the base.

52. The AST box of claim 43, wherein the plurality of reaction wells are configured to fit into corresponding wells in a temperature control block, wherein the temperature control block heats the plurality of reaction wells.

53. The AST cassette of claim 43, wherein the base, the diaphragm, and the cover each include an opening in the center of the AST cassette, the opening being compatible with being inserted by a pipette to move the AST cassette.

54. The AST cartridge of claim 43, wherein the diaphragm is overmolded into the cover to form a composite component, and wherein the composite component is assembled on the base by at least one of a snap fit connection or a mechanical fastener.

55. The AST box of claim 43, wherein the diaphragm is a single body extending across the plurality of reaction wells.

56. The AST box of claim 43, wherein the diaphragm comprises a plurality of parts assembled together, wherein each part covers each reaction well.

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

58. The AST cartridge of claim 43, wherein the diaphragm comprises a dual layer of polytetrafluoroethylene (PTFE) and another material selected from the group consisting of silicone, rubber, and butyl rubber.

59. The AST cartridge of claim 43, wherein the diaphragm comprises a thickness of about 1 to 2 mm.

60. The AST box of claim 43, wherein the plurality of reaction wells comprises about 100 reaction wells, wherein each reaction well holds a volume of about 30 microliters.

61. The AST box according to claim 43, wherein the cover is made of polypropylene (PP) or polycarbonate (PC) material.

62. The AST box according to claim 43, wherein the base is made of polystyrene (PS) material.

63. The AST cartridge of claim 43, wherein the cover includes an identifier that is scanned by an analyzer device for antimicrobial susceptibility testing.

64. The AST box of claim 63, wherein the identifier is a data matrix or a bar code.

65. A system for enriching a sample, the system comprising: a housing configured to receive a sample container containing a sample containing a pathogen; a pipette system, the pipette system being arranged inside the housing; one or more centrifuges, the one or more centrifuges being disposed within the housing; as well as A controller, wherein the controller is configured to: transferring at least a portion of the sample from the sample container to a processing tube using the pipette system; centrifuging the processing tubes using the one or more centrifuges to concentrate pathogens in the transferred samples; removing fluid from the processing tube using the pipette system, leaving concentrated pathogens in the processing tube; adding growth medium to the concentrated pathogens in the process tube using the pipette system to grow the concentrated pathogens in the process tube for a predetermined period of time; and The concentrated pathogens are cleaned after the predetermined period of time to obtain an enriched sample in the processing tube.

66. The system of claim 65, wherein the sample container is a blood culture bottle in which the sample is incubated for an amount of time to allow the pathogen to grow but not reach a growth plateau.

67. The system of claim 65, wherein the sample container is a blood sample tube.

68. The system of claim 65, further comprising a mixer disposed in the housing, wherein the sample is a blood sample, and wherein the controller is further configured to, prior to centrifuging the processing tube: add one or more lysis reagents to the processing tube by using the pipette system; The one or more lysing reagents are mixed with the blood sample in the processing tube using the mixer to lyse blood cells in the blood sample.

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

70. The system of claim 65, wherein the cleaning of the concentrated pathogens comprises centrifuging the processing tubes using the one or more centrifuges and removing the supernatant from the processing tubes using the pipette system to leave an enriched sample in the processing tubes.

71. The system of claim 65, further comprising an antimicrobial susceptibility testing (AST) subsystem, wherein the processing tube further comprises magnetic beads configured to attach to concentrated pathogens in the processing tube, and wherein the AST subsystem is configured to: applying 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 use the pipette system to remove extraneous liquid from the processing tube, thereby generating an enriched sample having the concentrated pathogen.

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

73. The system of claim 71, wherein the magnetic beads are coated with a specific ligand that is specific for a particular pathogen of the concentrated pathogens in the processing tube.

74. The system of claim 65, wherein the controller is further configured to: One or more wash materials are added to the concentrated pathogens in the processing tube using the pipette system to clean and remove any blood components or debris from the concentrated pathogens, leaving an enriched sample in the processing tube.

75. The system of claim 74, wherein the one or more washing materials comprise a combination of one or more buffers, detergents, surfactants, and proteases.

76. The system of claim 65, wherein the sample container and the processing tube each comprise a septum that permits insertion of a first needle coupled to a first pipette in the pipette system.

77. A system according to claim 76, wherein transferring at least a portion of the sample from the sample container to the processing tube includes inserting the first needle through the septum of the processing tube and dispensing at least a portion of the sample into the processing tube through the first needle.

78. The system of claim 65, further comprising: a receiving container configured to receive an antimicrobial susceptibility test (AST) cartridge containing a plurality of reaction wells; as well as A microscope configured to acquire one or more images of concentrated pathogens in the enriched sample after transferring the enriched sample to the plurality of reaction wells.

79. The system of claim 78, wherein the one or more centrifuges comprise: a first centrifuge configured to hold and centrifuge the processing tube; as well as A second centrifuge is configured to hold and centrifuge the AST cartridge in a vertical orientation.

80. The system of claim 78, further comprising: A magnet station is configured to apply a magnetic force to move the concentrated pathogen to a bottom surface of a reaction well in the AST cartridge, wherein the concentrated pathogen is attached to magnetic beads.

81. A system for analyzing a sample, the system comprising: a housing configured to receive a processing tube and an antimicrobial susceptibility test (AST) cartridge, the AST cartridge comprising: a plurality of reaction wells, each reaction well comprising a bottom wall, wherein the bottom wall is optically transparent; a heater, the heater being arranged inside the housing; a pipette system, the pipette system being arranged inside the housing; a microscope disposed inside the housing; and A controller, wherein the controller is configured to: dispensing a plurality of aliquots of the enriched sample containing the pathogen from the processing tube to a plurality of reaction wells in the AST cartridge using the pipette system, wherein each aliquot corresponds to a respective reaction well, and wherein each reaction well contains a predetermined concentration of an antimicrobial agent; incubating the aliquots in the reaction wells of the AST cartridge for a predetermined period of time using the heater to allow a reaction to occur between the pathogen and the antimicrobial agent in each reaction well; Using the microscope, acquiring one or more images of the bottom wall of each reaction well in the AST box; and The one or more images are analyzed by a processor coupled to the microscope to determine the susceptibility of the pathogen to the antimicrobial agent in each corresponding reaction well.

82. The system of claim 81 , wherein the controller is further configured to: Prior to acquiring the one or more images, a first fluorescent dye is added to each reaction well using the pipetting system to stain pathogens in an aliquot of the reaction well.

83. The system of claim 82, wherein the acquiring of the one or more images comprises acquiring one or more fluorescent images to detect a first fluorescent dye in each reaction well of the AST box.

84. The system of claim 83, wherein the analysis of the one or more images comprises calculating the number of fluorescent pathogens in each reaction well in the AST box.

85. The system of claim 82, wherein the controller is further configured to: Prior to acquiring the one or more images, a second fluorescent dye is added to each reaction well using the pipetting system.

86. A system according to claim 85, wherein the first fluorescent dye comprises a DNA binding dye that marks live cells in the aliquot of the reaction well, and the second fluorescent dye comprises a fluorescent intercalator that marks dead cells in the aliquot of the reaction well.

87. A method of making an antimicrobial susceptibility test (AST) kit, the method comprising: manufacturing a cover member including a plurality of openings; overmolding a diaphragm into the cap, wherein a first side of the diaphragm extends across the plurality of openings; generating a base comprising a plurality of reaction wells; as well as The base is attached to the second side of the diaphragm, wherein the second side of the diaphragm extends across and seals a plurality of reaction wells in the base.

88. The method of claim 87, wherein the cover comprises polypropylene (PP) or polycarbonate (PC).

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

90. The method of claim 87, further comprising: adding a predetermined concentration of an antimicrobial agent in liquid form to each reaction well prior to attaching the base to the second side of the membrane; as well as The antimicrobial agent in each reaction well is dried to a dry form or a freeze-dried form using forced air.

91. The method of claim 90, wherein the addition of the antimicrobial agent in each reaction well and the drying of the antimicrobial agent are completed within a predetermined time period to prevent degradation of the antimicrobial agent.

92. The method of claim 91, wherein the predetermined period of time comprises approximately 15 minutes.

93. The method of claim 91, wherein the predetermined period of time comprises approximately 10 minutes.

94. The method of claim 87, wherein manufacturing of the cover comprises using injection molding.

95. The method of claim 87, wherein attaching the base to the second side of the diaphragm comprises using at least one of a snap-fit ​​connector or a mechanical fastener.

96. The method of claim 87, further comprising: During attachment of the base to the second side of the diaphragm, each opening in the cover is aligned with a corresponding reaction well of the plurality of reaction wells in the base.

97. A method according to claim 87, wherein the generation of the base includes generating the plurality of reaction holes connected together as a single component.

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

99. The method of claim 87, wherein the diaphragm comprises a double layer of polytetrafluoroethylene (PTFE) and another material selected from the group consisting of silicone, rubber, and butyl rubber.

Images