WO2022231651A1

WO2022231651A1 - Magnetic mixer and methods to improve microbial time to detection in calorimetry

Info

Publication number: WO2022231651A1
Application number: PCT/US2021/055195
Authority: WO
Inventors: Kristin BAKER; Mark Somers; Kristen Roberts; Eric Stern
Original assignee: SeLux Diagnostics, Inc.
Priority date: 2021-04-30
Filing date: 2021-10-15
Publication date: 2022-11-03

Abstract

This application relates to clinical microbiology systems and methods, particularly devices, systems and methods providing improved mixing methods for analysis of whole blood samples within a calorimeter 430 without contamination.

Description

MAGNETIC MIXER AND METHODS TO IMPROVE MICROBIAL TIME TO DETECTION IN CALORIMETRY

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority under 35 USC § 119 to United States Provisional Patent Application Serial No. 63/182,410, filed April 30, 2021, which is incorporated by reference herein in its entirety and for all purposes.

FIELD OF THE DISCLOSURE

[0002] This application relates to clinical microbiology systems and methods, particularly devices, systems and methods providing improved mixing methods for analysis of whole blood samples within a calorimeter without contamination.

BACKGROUND OF THE DISCLOSURE

[0003] Antimicrobial susceptibility tests (AST) evaluate the effectiveness of antimicrobial drugs against microorganisms found in samples obtained from patients and provide a route to personalized, targeted treatment regimens. Often such samples come from blood samples which need to be cultured to allow sufficient microbial growth for testing, detection and evaluation before a clinical decision can be made. One culturing method in current clinical microbiology laboratories employs continuous monitoring blood culture systems which are readily automated. Such automated culturing systems improve reproducibility, minimize contamination and speed time to results. Further, continuous monitoring blood culture systems are (1) cost effective, using generally low-cost consumables with minimal need for human intervention, and (2) provide positive predictive values (PPVs).

[0004] Continuous monitoring blood culture systems suffer three primary shortcomings that may delay results. First, these systems require bacteria to grow in the presence of bloodborne agents that may inhibit bacterial growth, such as white blood cells and platelets, cationic peptides, and intravenous antibiotics. Second, because of the CC -based detection mechanisms employed by the leading systems, positive results are often not obtained until bacterial levels of 10⁷-10⁸ CFU/mL are achieved. Third, since continuous monitoring is required by the detection modalities, in a consolidated healthcare system where bottles can be collected at sites that are geographically separated from the central clinical laboratory, incubation of the bottles cannot commence until they are loaded into the central laboratory’s continuous monitoring platform. Thus, time waiting and in transport may be lost, as a plurality of culture growth time must be in the system. In addition to being a significant disadvantage for integrated health networks, the inability to incubate samples outside the laboratory may also slow results in large hospitals, where bottles may not be immediately transported to laboratories upon being filled.

[0005] Calorimetry is a highly sensitive method for detecting changes in heat and heat flow, and in medical diagnostics, calorimetry may offer superior sensitive detection of cellular respiration to aid diagnosis in a wide-ranging array of diseases, including sepsis. Calorimetry has recently been shown to be capable of detecting blood culture positive microbial growth faster than other methods.

[0006] Microbial growth can be detected by isothermal calorimetry through the heat generated by microbial metabolic activities. For example, the positive heat flow generated by microbial activity can be differentiated from the negative heat flow associated with blood in the presence of anticoagulants, such as sodium polyanethole sulfonate (SPS), the standard anticoagulant used for blood culture and growth media.

[0007] Because the heat flows from microbial growth and blood background run in opposing directions and given the high sensitivity of calorimeters, early detection of microbial growth with isothermal calorimetry is possible and practical - nonetheless, improvements are still needed that can reduce the time to detection (TTD), for example, as would be obtained if microbial growth rates can be increased. One method to improve microbial growth rates is mixing of the blood culture sample during monitoring the heat flow associated with microbial growth. For some organisms, such as obligate aerobes and non-motile organisms, mixing may be needed for sufficient growth to ensure adequate heat flows for faster detection. However, mixing blood culture samples during isothermal calorimetry measurements presents multiple challenges, including maintaining sterility conditions without excessive signal noise.

[0008] Standard calorimeter detectors reside at the base of the calorimetry vessel, and any heat generated from stirring at this level may affect the heat flow signal more greatly than for disturbances generated at the top of the vessel. Thus, frictional heat generation and/or vibration may obliviate the practicality of using any “stir plate” style mixers where the stirring apparatus resides on the base of the vessel. Further, current overhead calorimetry mixers, such as the TAMAir Admixer, are not feasible for use as they can cause sample contamination.

[0009] Accordingly, there remains a need for sample mixing systems and methods that reduce the risk of contamination and more simply and quickly detect microbial growth when compared with conventional methods.

SUMMARY OF THE DISCLOSURE

[0010] In an aspect of the present disclosure, a system for mixing a blood culture sample or other sample, particularly samples that need to maintain sterility and/or avoid contamination, may include a mixer comprising a first end, a second end, and a longitudinal axis therethrough.

A mixer magnet may be coupled to the mixer along the longitudinal axis. A sealed vessel, including a hermetically sealed vessel, may contain the mixer. A drive shaft may be spaced a distance away from the mixer. The drive shaft may extend substantially axially along the longitudinal axis and may be rotatable about the longitudinal axis. A drive magnet may be coupled to the drive shaft.

[0011] In various embodiments of the present disclosure, the mixer may be magnetically suspended such that the mixer is contactless with at least a bottom surface of the vessel and/or a drive shaft. The drive magnet may be coupled to the drive shaft via a radial member extending radially away from the drive shaft. A scaffold may be rotatably coupled to the mixer. The scaffold may extend about a mixing portion of the mixer. The mixer may comprise at least one radial member extending radially away from the longitudinal axis. At least one of the mixer magnet and the drive magnet may comprise a diametric magnet. The mixer may comprise one of a rod, a radial extension, a projection, an impeller, a propeller, a turbine, a fan, a helix and the like. The mixer may be over molded with the mixer magnet. A scaffold may be coupled to a top portion of the vessel. The mixer may be rotatably coupled to the scaffold

[0012] In an aspect of the present disclosure, a mixing device for mixing a sample may include a vessel comprising a reservoir. A magnetic mixer may be disposed within the reservoir. The magnetic mixer may comprise a mixer comprising (a) a first end, a second end, and a longitudinal axis therethrough and (b) a magnet coupled to the mixer along the longitudinal axis. A cap may be coupled to the vessel. The cap may comprise a septum. Magnet mixers of the disclosure include, but are not limited to, any mixer described herein that can be disposed within a vessel with any of the embodiments or aspects of mixers as described herein.

[0013] In various embodiments of the present disclosure, a scaffold may be rotatably coupled to the mixer. The scaffold may extend about a mixing portion of the mixer. The magnet may comprise a diametric magnet. The mixer may comprise at least one radial member extending radially away from the longitudinal axis. The mixer may be over molded with the magnet. A scaffold may be coupled to a top portion of the vessel. The mixer may be rotatably coupled to the scaffold. The mixer may comprise one of a rod, a radial extension, a projection, an impeller, a propeller, a turbine, a fan, a helix and the like.

[0014] In an aspect of the present disclosure, a method of mixing a sample, including but not limited to, blood culture samples or other samples that need to maintain sterility, comprises transferring to or directly collecting the sample into a sealed vessel, and preferably a hermetically- sealed vessel; and suspending and rotating the mixer within the vessel such that the mixer is contactless with at least one surface of the vessel, e.g., the sides and bottom of the vessel, and measuring a metric of the sample. In an embodiment, a drive shaft is contactless with the vessel. In an embodiment, the mixer may be suspended and in contact with a top, lid or cap of the vessel. A cap may include a recess at a side of the cap oriented toward a reservoir of the vessel. Measuring a metric of the sample, includes but is not limited to, the heat flow of the blood culture sample during culturing or incubation of such a sample, or measuring the heat flows in other samples. A scaffold may be coupled to a top portion of the vessel. The mixer may be rotatably coupled to the scaffold

[0015] In various embodiments of the present disclosure, suspending the mixer may further comprise placing the vessel in axial alignment with a drive magnet. Rotating the mixer may further comprise rotating the drive magnet. The mixer may further comprise a scaffold rotatably coupled to the mixer. The scaffold may extend about a mixing portion of the mixer. Measuring a metric of the blood culture sample may further comprise disposing the vessel within a calorimeter. The vessel may be suspended within the calorimeter such that the vessel is contactless with a surface of the calorimeter. Rotating the mixer may further comprise a contactless magnetic coupling of a drive shaft magnet with a mixer magnet.

[0016] In an aspect of the present disclosure, a device for mixing liquid samples within a sealed vessel within a calorimeter may include a mixer contained within the vessel capable of moving liquid, resuspending sedimented material, and/or combining separate components within the vessel when rotated or otherwise moved with a magnet external to the vessel.

[0017] In various embodiments of the present disclosure, a shaft capable of rotating or otherwise moving may be external to and physically above the vessel that comprises a magnet capable of rotating or otherwise moving and magnetically coupling to the mixer. The mixer may not contact the bottom surface of the vessel. The mixer may not rest on the bottom surface of the vessel. The mixer may comprise one or more magnets. The mixer may comprise one or more magnetically addressable material. One or more magnets may comprise neodymium. The magnet and/or magnetically addressable material of the mixer may be enclosed within an inert casing. A majority of the mixer may be plastic. A portion of the mixer may be flexible. The flexible portion of the mixer may be compressed to be inserted through the opening of the vessel. The mixer diameter may be within about 10%, 25%, 50%, 60%, 70%, 80%, or 90% of the inner diameter of the vessel. The mixer height may be within about 10%, 25%, 50%, 60%, 70%, 80%, 90% of the height of the vessel. The mixer may comprise one or more openings. The shaft may be connected to an electric motor. The electric motor may be outside the calorimeter. The shaft diameter may be less than about 5 mm. The magnet may be connected to the shaft at a distance greater than about 5 mm from the center of the shaft. The shaft may comprise two or more magnets. The magnet may be connected to the shaft within about 5 mm, 10 mm, 15 mm, 20 mm, 25 mm, 30 mm, 40 mm, or 50 mm of the top of the vessel. The mixer may be contained within a flexible scaffold that suspends the mixer above the bottom of the vessel. The mixer may float within the sample, e.g., as it might when the vessel contains growth media or other liquid.

[0018] In an aspect of the present disclosure, a device for mixing liquid samples within a sealed vessel outside of a calorimeter for subsequent detection within a calorimeter may include a mixer contained within the vessel capable of moving liquid or resuspending sedimented material or combining separate components within the vessel when rotated or otherwise moved with a magnet external to the vessel.

[0019] In various embodiments of the present disclosure, the magnet may be external to the vessel may be either above or below the vessel. A shaft may be capable of rotating or otherwise moving external to and above the vessel. The shaft may comprise a magnet capable of rotating or otherwise moving and magnetically coupling to the mixer. The top and the bottom of the mixer residing within the vessel may be magnetic. The device and/or other components are partially or entirely enclosed within a calorimeter or within a temperature control thermostat. The device may be used in either a top-loading or a side-loading calorimeter.

[0020] In an embodiment, shielding of the magnetic material from other samples may be advantageous in a multi-channel calorimeter. Interference from magnetic mixing in adjacent channels may compromise mixing and contribute to signal noise within the calorimeter or less efficient mixing. Magnetic shielding materials can consist of any material that reduces, redirects, or absorbs magnetic flux between a magnet’s north and south poles. Typically, such materials include ferromagnetic alloys. While such materials are typically iron-based, other useful shielding materials, include but are not limited to, nickel, molybdenum, manganese, cobalt, copper, chromium, magnetic oxides, silicon, and carbon. In an embodiment, a conductive shielding connected to an electrical ground reference either directly or through a tuned filter network may be used to shield magnetic interference between channels of the calorimeter. Alternatively, and also in addition, the sample channel spacing can be altered to reduce magnetic interference between channels.

[0021] In another embodiment, a battery-driven motor residing within the sample vessel can drive the mixer of the disclosure. In this regard, the mixing may occur within the sample vessel and be driven by a battery residing within the lid of a hermetically sealed sterile vessel, or the lid of any other sealed vessel. By way of example, mixing can be initiated through a mechanical mechanism such as a twisting of a cap to physically turn the device on, a wireless connection such as infrared, Bluetooth, or encrypted radio frequency enabling mixing to be initiated or via a timer function.

[0022] Another aspect of the present disclosure is directed to methods of detecting microbial growth in a blood culture sample which comprises (a) incubating the blood culture sample under isothermal conditions for a time and at a temperature sufficient for microbial growth to occur, wherein the blood culture sample is obtained by aseptic transfer of blood into a sterile mixing device of the disclosure which has a contents comprises (i) media adapted for growing microorganisms in the presence of whole blood, and (ii) a magnetic mixer of the disclosure; (b) mixing the blood culture sample by magnetically engaging the magnetic mixer into a position within the mixing device that provides for mixing without the magnetic mixer contacting at least the bottom of the vessel portion of the device; and (c) monitoring the heat flow produced by the sample to determine whether microbial growth is occurring. [0023] A further aspect of the disclosure provides a system for mixing a sample which comprises (a) a mixer comprising a first end, a second end, and a longitudinal axis therethrough; (b) a motor coupled to the mixer along the longitudinal axis; (c) a battery powering the motor; and (d) a vessel containing the mixer, the motor, and the battery. In an embodiment of the system, the vessel is configured to incubate a blood culture sample under isothermal conditions for a time and at a temperature sufficient for microbial growth to occur, wherein the blood culture sample is obtained by aseptic transfer of blood, and wherein the vessel contains a media adapted for growing microorganisms in the presence of whole blood.

[0024] In an embodiment of the system, the vessel is hermetically sealed. In an embodiment of the system, the mixer is configured to engage at least one of a top of the vessel and a cap of the vessel. In other embodiments, the motor is configured to operate by one of a wireless signal and a mechanical switch. In an embodiment of the system the vessel is configured to incubate a blood culture sample under isothermal conditions suitable for detecting heat flow by monitoring via isothermal calorimetry. In an embodiment of the system, the vessel is configured to incubate a blood culture sample under isothermal conditions suitable for detecting microbial growth. In an embodiment of the system, the mixer is configured for use in an automated platform system. In an embodiment, a scaffold is coupled with the vessel. In an embodiment, the mixer is rotatably coupled to the scaffold.

[0025] It is to be understood that while the mixing devices and systems described herein refer to devices and systems for mixing blood culture samples, these devices and systems are not to be so limited and can be readily adapted to devices and systems for mixing fluids, powders and the like of any kind, especially for fluids, powders and the like which may be sealed or contained within a vessel where sterility should be maintained.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 shows heat flows measured from blood samples inoculated with Klebsiella pneumoniae under mixed (a) and non-mixed (b) conditions.

[0027] Figure 2A and Figure 2B show heat flows measured from uninoculated blood samples under mixed and non-mixed conditions, respectively. [0028] Figure 3 is a bar graph showing the TTD for blood samples inoculated with Candida auris or Candida tropicalis under mixed (black bars) or non-mixed (pink bars) conditions as determined from heat flows.

[0029] Figure 4 illustrates a system for mixing a blood culture sample within a calorimeter, according to an embodiment of the present disclosure.

[0030] Figure 5 illustrates a system for mixing a blood culture sample, according to an embodiment of the present disclosure.

[0031] Figure 6 illustrates a system for mixing a blood culture sample, according to an embodiment of the present disclosure.

[0032] Figure 7 illustrates a system for mixing a blood culture sample including a mixer over molded with a magnet, according to an embodiment of the present disclosure.

[0033] Figures 8A-8E illustrate various ends of a mixer, according to embodiments of the present disclosure.

[0034] Figure 9A is a photograph of a mixer foot with tape used to simulate drag across the bottom surface of a calorimetry vessel.

[0035] Figure 9B is a plot showing the continuous heat flow measured from the vessel in Fig. 9A with the mixer off, at low speed, off again, and at high speed. The inset is an expanded view of the heat flow during the period of low speed mixing.

[0036] Figure 10 shows heat flows measured from blood samples inoculated with Staphylococcus aureus using a magnetic mixer of the disclosure under (A) mixed conditions, (B) non-mixed conditions or for (C) an uninoculated, sterile control under non-mixed conditions. [0037] Figure 11 shows heat flows measured from blood samples inoculated with K. pneumoniae under mixing conditions using (A) a magnetic mixer of the disclosure or (B) a TAM Air Admixer.

[0038] Figure 12 illustrates a magnetic shielding arrangement in a multi-channel calorimeter. [0039] Figure 13 illustrates a mixer including a battery-driven motor.

[0040] Figure 14A illustrates a system for mixing a blood culture sample, according to an embodiment of the present disclosure.

[0041] Figure 14B illustrates a scaffold and a magnet of the system for mixing a blood culture sample of Fig. 14A. [0042] Figure 14C illustrates another view of the system for mixing a blood culture sample of

Fig. 14A.

[0043] Figure 15A illustrates a system for mixing a blood culture sample, according to an embodiment of the present disclosure.

[0044] Figure 15B illustrates another view of the system for mixing a blood culture sample of

Fig. 15A.

[0045] Figure 16A illustrates a system for mixing a blood culture sample, according to an embodiment of the present disclosure.

[0046] Figure 16B illustrates another view of the system for mixing a blood culture sample of

Fig. 16A.

DETAILED DESCRIPTION OF THE DISCLOSURE Definitions

[0047] In order that the present disclosure may be more readily understood, certain terms are defined below. Additional definitions may be found within the detailed description of the disclosure.

[0048] Throughout this specification, the word “comprise” or variations such as “comprises” or “comprising” will be understood to imply the inclusion of a stated integer (or components) or group of integers (or components), but not the exclusion of any other integer (or components) or group of integers (or components).

[0049] The singular forms “a,” “an,” and “the” include the plurals unless the context clearly dictates otherwise.

[0050] The term “including” is used to mean “including but not limited to.” “Including” and “including but not limited to” are used interchangeably.

[0051] The terms “patient,” “subject,” and “individual” may be used interchangeably and refer to either a human or a non-human animal. These terms include mammals such as humans, primates, livestock animals ( e.g ., cows, pigs), companion animals (e.g., dogs, cats) and rodents ( e.g ., mice and rats).

[0052] The term “non-human mammal” means a mammal which is not a human and includes, but is not limited to, a mouse, rat, rabbit, pig, cow, sheep, goat, dog, primate, or other non-human mammals typically used in research. As used herein, “mammals” includes the foregoing non human mammals and humans.

[0053] As used herein, vessel, container, ampoule, bottle and the like are used interchangeably. [0054] As used herein, “heat” or “heat flow” refers to the output signal detection from a calorimeter. As used herein, “calorimeter” refers to any apparatus that measures an amount of heat involved in a chemical reaction or other process and produces an output signal indicative of that heat. This signal may arise from the whole blood, microorganisms within the sample, growth media, anticoagulant, physical manipulation of the sample as it resides within the calorimeter or any other signal resulting from the sample itself or other components of its container. Herein the terms, “heat” or “heat flow” may be used interchangeably when referring to the signal produced by a calorimeter. Typically, heat flow rate from blood samples will decrease over time, for example due to a decrease in metabolic heat production over time. In accordance with embodiments described herein, the inventors have found, under certain conditions, the expected decrease in heat flow rate from whole blood samples is decreased by microbial growth. In other words, under certain conditions, the decrease in expected heat flow is slowed by microbial growth such that the rate of decrease in heat flow is decreased, eliminated, or reversed for some period of time. In embodiments, measured heat flow of the sample over time is added to produce a resulting calculation of total heat or accumulation over time. Blood cells will generate a linear increase in heat over time while microbial growth will result in an exponential increased or acceleration of heat over time. Embodiments make use of this discovery to enable the rapid detection of microbial growth in methods not previously known.

Overview

[0055] Various embodiments described herein present one or more improvements to devices, systems, and/or methods for calorimetric detection of microorganism growth and for other uses set forth herein, including but not limited to, mixing samples during isothermal calorimetry processes.

[0056] However, traditional means of mixing samples, for example, overhead mixers, may present risk of sample contamination. External mixers can be introduced into an ampoule containing a sample, for example, from an open end. The sample then may be contaminated by a substance on the mixer itself, from an external exposure via the opening, or both. Various embodiments herein reduce sample contamination risk by using mixer designs which can remain hermetically sealed throughout calorimetric processes. Further mixing needs to be accomplished with minimal additional friction generating heat in a sample.

[0057] Accordingly, various embodiments described herein include mixers which reduce signal noise from friction between mixers and vessel surfaces. For example, various mixers may not interact with the vessel surface, and/or various mixers may control an interaction between the mixer and the vessel surface so as to reduce friction therebetween.

Mixer Devices and Systems

[0058] Referring to FIG. 4, an embodiment of a system for mixing a blood culture sample is illustrated within a measuring apparatus 430, e.g., a calorimeter. The measuring apparatus 430 may be used to measure a metric of a blood culture sample contained within a hermetically sealed vessel 410, e.g., heat flow during sample growth, via a sensor of the measuring apparatus 430. A mixer 400 is contained within a reservoir of the vessel 410. The mixer 400 has a first end 400p, a second end 400d, and a longitudinal axis L therethrough. The second end 400d of the mixer 400 is a mixing portion of the mixer 400, which includes radial members 404 extending radially away from the longitudinal axis L that are configured to mix the sample within the vessel 410. A mixer magnet 402 is coupled to the first end 400p of the mixer 400 along the longitudinal axis L.

[0059] A drive shaft 422 extends axially along and is rotatable about the longitudinal axis L. The shaft 422 extends through a stationary sheath 426 extending partially along a length of the shaft 422 and is mounted to the measuring apparatus 430 through a cap 428. A heat sink 424 is disposed about the sheath 426 that may assist with reducing heat along the portion of the system away from the vessel 410. The sheath 426 extends from an exterior of measuring apparatus 430 into an interior thereof. The sheath 426 and cap 428 maintain a closed environment of the interior of the measuring apparatus 430. An end of the drive shaft 422 positioned towards the vessel 410 includes a drive magnet 420. This end of the drive shaft 422 is spaced a distance x away from the mixer 400. The distance x is such that there is a magnetic field interaction between the drive magnet 420 and the mixer magnet 402. The distance x may be set such that the mixer magnet 402 is magnetically attracted toward the drive magnet 420 substantially along the longitudinal axis L. This magnetic attraction of the mixer magnet 402 at the first end 400p may move the mixer 400 out of contact with a surface of the reservoir of the vessel 410. Throughout this disclosure, this configuration of the mixer 400 with the mixer magnet 402 moving the mixing portion of the second end 400d of the mixer 400 out of contact with at least one surface of the vessel may be considered “suspended”. In the suspended configuration, the mixer 400 may be actuated with rotation of the drive magnet 420 via rotation of the drive shaft 422 via a motor 430. Rotation of the drive magnet 420 about the longitudinal axis L magnetically rotates the mixer magnet 402 and mixer 400.

[0060] Also illustrated in FIG. 4 is a scaffold 412 within the vessel 410. The scaffold 412 is rotatably coupled to the mixer 400 such that the mixer 400 is free to rotate with respect to the scaffold 412. The scaffold 412 may have one or more dimensions, e.g., width, diameter, height, etc., that are larger than that of the mixer 400 such that the scaffold 412 may rest in contact with one or more surfaces of the reservoir of the vessel 410, e.g., a side of the vessel 410, with the mixer 400 remaining suspended, contactless with one or more surfaces of the vessel 410. The scaffold 412 may not significantly agitate the vessel 410 during rotational operation of the mixer 400 such that friction, heat, noise, or the like may be reduced. The scaffold 412 may assist with or replace the function of the drive magnet 420 suspending the mixer 400. The scaffold 412 may assist with centering the mixer 400 along the longitudinal axis L.

[0061] Systems and methods described herein may advantageously reduce contact between a mixer and a vessel. Reduced contact between a mixer and a vessel may reduce friction between the two. Reduced friction may reduce vibrations and/or heat affecting a sample and/or a sensor. Undesirable friction in a mixing system may increase errant data (“noise”) read by a sensor and produced by a measuring apparatus. Therefore, reduced system friction may allow for increased sensor sensitivity and more accurate data. For example, a mixer frictionally contacting a vessel surface in operation (i.e., scraping, tapping, rubbing, dragging, or the like) may generate frictional heat read by a sensor, producing background noise in output data, reducing sensitivity and accuracy of the system. Friction is a greater concern in proximity to a sensor. For example, in systems where a sensor resides at a base of a vessel, mixer and vessel frictional contact is of greater concern towards the base than towards the top of the vessel. Mixers described herein may include dimensions smaller than internal dimensions of a vessel containing the mixer such that frictional contact is reduced. [0062] A mixer herein may be dimensioned to substantially extend throughout an internal dimension of a vessel and/or a dimension of a sample volume. A mixer extending substantially across a dimension of a blood culture sample volume may allow for thorough mixing of the sample compared to a mixer extending significantly less than across a dimension of a blood culture sample volume. A mixer sized to a blood culture sample volume may reduce the size of portions of the blood culture sample volume that are not mixed by the mixer, i.e., minimizing “dead zones” in which sample mixing is not achieved. For example, a mixer may extend across a majority of one or more dimensions of a sample or vessel volume such as a height and/or a diameter. For example, a mixer may extend about 50%, 60%, 70%, 80%, 90%, 95%, or the like of a height and/or a diameter of a vessel. A mixer may be any shape, e.g., a rod, a radial extension, a plane, a projection, an impeller, a propeller, a turbine, a fan, a helix, a combination thereof, or the like. A mixer may be symmetrical or asymmetrical about a longitudinal axis along its length. A mixer may have a substantially uniform dimension or a variable dimension throughout. A mixer may have a solid body or apertures or channels throughout. A mixer may be flexible or rigid as desired. A flexible mixer may advantageously be insertable through a narrow opening of a vessel by constraining to a smaller volume until the mixer is deployed into a reservoir of the vessel where it may expand to a larger volume.

[0063] In various embodiments, a mixer may rotate about a central axis, e.g., a longitudinal axis extending axially through the mixer. Rotation may by driven by contactless magnetic interaction with a rotating external drive shaft including a magnet. The drive shaft may be suspended a distance away from a mixer and/or vessel that may be about 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 10 mm, 15 mm, 20 mm, 25 mm, 30 mm, 40 mm, 50 mm, or the like. A drive shaft may extend outside a measuring apparatus, e.g., outside of a thermal equilibration zone of a calorimeter. A motor may rotate a drive shaft externally from the measuring apparatus. A drive shaft may vary in diameter, e.g., about 0.5 mm, 1, mm, 2 mm, 3 mm, 4 mm, 5 mm, or the like. A drive shaft may have a variable diameter along its length such that one or more magnets may be coupled to the shaft such that the overall diameter of the drive shaft including the one or more magnets has a substantially uniform outer diameter along a length of the shaft. A drive shaft may include multiple parts, e.g., a stationary outer tubular member or sheath and a rotating internal rod. Dimensions of shafts, mixers, magnets, vessels, and distances between them may be adjusted to accommodate functional needs of a system. For example, a distance between a magnet of a drive shaft and a magnet of a mixer may be adjusted such that the mixer is suspended away from a surface of a vessel and/or such that a vessel is suspended away from a surface of a measuring apparatus such as a calorimeter. In order to suspend a vessel away from a surface, e.g., a bottom, a stage, or the like, of a measuring apparatus a mixer magnet may be moved towards a drive shaft magnet across a cap or top of the vessel such that the cap or top of the vessel is also moved toward the drive shaft.

[0064] In various embodiments described herein, one or more magnets of a shaft and/or mixer may be axial or diametric magnets. Diametric magnets may have radial magnetic poles rather than axially magnetic poles. A magnet herein may be any shape, for example, a solid cylinder, a hollow cylinder, a solid disk, a hollow disk, or the like. A magnet herein may be adhered to or otherwise formed with a shaft or mixer. A coating may be applied to one or more magnets that may be inert to reduce undesirable interaction between a magnetic surface and a sample of the system. A magnet herein may comprise numerous materials, e.g., neodymium or the like. A magnet herein may be uniformly magnetized, nonuniformly magnetized, or comprise one or more magnetic portions implanted in an otherwise non-magnetic material.

[0065] In various embodiments, a mixer may be freely suspended within a vessel or a mixer may be rotatably coupled to a cap of a vessel. A mixer coupled to a cap of a vessel may be spaced away from one or more surfaces, e.g., bottom, side, or the like, and be free to rotate about its longitudinal axis. A side of a cap oriented toward an a reservoir of a vessel may include a feature to accept and/or center a mixer, e.g., a deformation, a cavity, a notch, a dent, or the like, being magnetically manipulated by a drive magnet. A cap may include an axle for a mixer and/or a mixer magnet to rotatably couple to it such that a drive magnet may move (i.e., lift) the mixer magnet and cap, and also rotate the mixer magnet while the cap remains substantially stationary. Alternatively, a mixer and/or mixer magnet may be coupled to a crimp top or handling hook of a vessel.

[0066] In various embodiments, a mixer may be suspended within a scaffold. The mixer may rotate independently of the scaffold. A scaffold may float within a sample of a vessel. A scaffold may space a mixer therein away from a surface of a vessel.

[0067] In various embodiments, mixing may be performed external or internal of a measuring apparatus. Mixing may include rotation of a mixer and/or agitation of a sample, e.g., by physical rocking, rotational movement, or inversion of a vessel. [0068] Referring to FIG. 5, a system for mixing a blood culture sample is illustrated according to an embodiment of the disclosure including a mixer 500. The mixer 500 is contained within a reservoir of a vessel 510. The mixer 500 has a first end 500p, a second end 500d, and a longitudinal axis L therethrough. The mixer 500 includes multiple radial extension members 504 extending radially away from the longitudinal axis L and also extending axially along a length of the mixer 500 from the second end 500d towards the first end 500p that are configured to mix a sample. A mixer magnet 502 is coupled to the first end 500p of the mixer 500 along the longitudinal axis L.

[0069] A drive shaft 522 extends axially along and is rotatable about the longitudinal axis L. The drive shaft 522 includes a jog 524 that extends radially away from the longitudinal axis L and then substantially parallel with the longitudinal axis L to a drive magnet 520 at an end of the shaft 522. The jog 524 extends the magnet 520 a distance y away from the longitudinal axis L for manipulating the mixer 500. The distance y is such that there is a magnetic field interaction between the drive magnet 520 and the mixer magnet 502. The distance y may be set such that the mixer magnet 502 is magnetically attracted toward the drive magnet 520 substantially perpendicular to the longitudinal axis L. This magnetic attraction of the mixer magnet 502 at the first end 500p may rotate the mixer 500 about the longitudinal axis L within the vessel 510 as the drive magnet 520 revolves about the longitudinal axis L with rotation of the drive shaft 522. [0070] As illustrated in FIG. 5, the mixer 500 includes radial extensions 504 that are tapered to a smaller outer diameter at the first and second ends 500p, 500d of the mixer 500. These tapered ends of the radial extensions 504 may reduce frictional contact between the radial extensions 504 and the vessel 510. The radial extensions 504 of the mixer 500 are arrayed about the longitudinal axis L and fill a substantial portion of the reservoir dimensions of the vessel 510. For example, the outer diameter D500 of the mixer 500 is similar to but smaller than the diameter D510 of the reservoir of the vessel 510 and the height H500 of the mixer 500 is similar to but smaller than the height H510 of the reservoir of the vessel 510.

[0071] Referring to FIG. 6, a system for mixing a blood culture sample is illustrated according to an embodiment of the disclosure including a mixer 600. The mixer 600 is contained within a reservoir of a vessel 610. The mixer 600 includes a first end 600p, a second end 600d, and a longitudinal axis L therethrough. The mixer 600 includes a radial extension member 604 extending radially away from the longitudinal axis L and also extending axially along a length of the mixer 600 from the second end 600d towards the first end 600p that is configured to mix a sample. The radial extension member 604 extends helically about the longitudinal axis L and has a larger outer dimension at the second end 600d than at the first end 600p. The larger second end 600d may increase mixing towards the bottom of the reservoir of the vessel 610 where a blood culture sample may reside. A mixer magnet 602 is coupled to the first end 600p of the mixer 600 along the longitudinal axis L.

[0072] A drive shaft 622 extends axially along and is rotatable about the longitudinal axis L. An end of the drive shaft 622 positioned towards the vessel 610 includes a drive magnet 620.

This end of the drive shaft 622 is spaced a distance away from the mixer 600. The distance is such that there is a magnetic field interaction between the drive magnet 620 and the mixer magnet 602. The distance may be set such that the mixer magnet 602 is magnetically attracted toward the drive magnet 620 substantially along the longitudinal axis L. This magnetic attraction may suspend the mixer 600 such that it is contactless with one or more surfaces of the vessel 610. The magnetic attraction of the mixer magnet 602 allows for the drive magnet 620 to rotationally operate the mixer 600 via shaft 622 rotation about the longitudinal axis L. The vessel 610 includes a cap 606 for hermetically sealing the vessel 610. The cap 606 may include a septum for inserting a sample into the hermetically sealed vessel 610 containing the mixer 600. [0073] Referring to FIG. 7, a system for mixing a blood culture sample is illustrated according to an embodiment of the disclosure including a mixer 700. The mixer 700 is contained within a reservoir of a vessel 710. The mixer 700 includes a first end 700p, a second end 700d, and a longitudinal axis L therethrough. The mixer 700 includes a radial extension member 704 extending radially away from the longitudinal axis L that is configured to mix a sample. The radial extension member 704 proportionally occupies a lower percentage of volume of the reservoir of the vessel 710 compared to some other embodiments described herein such that the member 704 may mix a sample with minimal agitation. A mixer magnet 702 is coupled to the first end 700p of the mixer 700 along the longitudinal axis L. The mixer 700 may be over molded with the mixer magnet 702 along at least a portion 700m of the mixer 700.

[0074] A drive shaft 722 extends axially along and is rotatable about the longitudinal axis L. An end of the drive shaft 722 positioned towards the vessel 710 includes a drive magnet 720 encased by an insulative barrier 728, e.g., a polymer layer, a rubber layer, or the like, that may assist with protecting the drive magnet 720 and drive shaft 722 from errant collisions due to, e.g., magnetic attraction with system parts. The end of the drive shaft 722 is spaced a distance away from the mixer 700. The distance is such that there is a magnetic field interaction between the drive magnet 720 and the mixer magnet 702 for the mixer magnet 702 to be magnetically attracted toward the drive magnet 720 substantially along the longitudinal axis L. This magnetic attraction may suspend the mixer 700 such that it is contactless with one or more surfaces of the vessel 710. The magnetic attraction of the mixer magnet 702 allows for the drive magnet 720 to rotationally operate the mixer 700 via shaft 722 rotation about the longitudinal axis L.

[0075] With reference to FIGS. 8A-8E, various ends 800d of mixer 800 embodiments are illustrated, according to the present disclosure. Each mixer 800 includes a first end 800p, a second end 800d, and a longitudinal axis L therethrough. The first end 800p of each mixer 800 includes a mixer magnet 802 that is substantially aligned with the longitudinal axis L of the mixer so that the mixer magnet 802 may be magnetically manipulated by a drive magnet as described herein. The second end 800d of each mixer 800 includes one or more radial extension member 804a, 804b, 804c, 804d, 804e extending radially away from the longitudinal axis L. The radial extension member 804a, 804b, 804c, 804d, 804e of each mixer is at the second end 800d of each mixer 800 such that the second end 800d may be oriented toward a bottom portion of a vessel where a significant volume of a blood culture sample may reside for mixing. Various radial extension members 804a, 804b, 804c, 804d, 804e may be desirable to increase or decrease mixing characteristics depending on or related to, e.g., sample viscosity, mixing time, growth rate, virus or bacteria potency, heat generation, friction generation, sample volume, reservoir volume of a vessel, mass of a mixer, mass of a mixer magnet, mass of a driver magnet, mixer drag forces, or the like.

[0076] Referring to FIG. 8A, extension members 804a may be planar or cylindrical members extending radially from and symmetrically about the longitudinal axis L. Referring to FIG. 8B, extension member 804b may be a planar or a cylindrical member extending radially from the longitudinal axis L and is asymmetrical about the longitudinal axis L. Referring to FIG. 8C, extension members 804c may be planar or cylindrical members with a first portion 806 extending radially from and symmetrically about the longitudinal axis L with connector portions 808 extending parallel to the longitudinal axis L and connecting the first portions 806. Filler portions 812 within the perimeter of first portions 806 and the connector portions 808 may be spaced to establish agitation edges for mixing. Referring to FIG. 8D, extension members 804D are cylindrical members extending radially from the longitudinal axis L and are asymmetrical about the longitudinal axis L. The extension members 804D are arranged axially along the longitudinal axis L and extend in the same general radial direction from the longitudinal axis L. The mixer magnet 802 of FIG. 8D is disposed within a vessel 810. The vessel 810 may be capped at a top 828 of the vessel and enclosed within a sterile bag 830 as an exemplary packaging for delivery and storage. Referring to FIG. 8E, extension members 804e may be planar or cylindrical members extending radially from and symmetrically about the longitudinal axis L. Apertures 814 within the extension members 814 may establish agitation edges for mixing. Similar apertures 814 may be applied to other mixer embodiments.

[0077] With reference to FIG. 12, a series of systems 1250 for mixing a sample similar to that as described and illustrated with respect to FIG. 4 are illustrated within a multichannel calorimeter 1230, according to an embodiment of the present disclosure. The systems 1250 each include a drive magnet 1220 and a vessel 1210 containing a mixer 1200 as described herein. Between the systems 1250 are a magnetic shielding block 1260 to decrease or eliminate magnetic fields produced from drive magnets 1220 and/or mixer magnets 1210 from affect adjacent systems 1250 within the multichannel calorimeter 1230.

[0078] Referring to FIG. 13, a system for mixing a sample is illustrated including a vessel 1310 and a cap 1306 coupled to the vessel 1310, according to an embodiment of the present disclosure. The cap 1306 includes a battery-powered motor 1340 coupled to the cap 1306. The motor 1340 is coupled to a mixer 1300 such that the mixer 1300 may be rotated about a longitudinal axis L. The mixer 1300 may be operated without a driving force external of the vessel 1310. Internal parts of the vessel 1310, e.g., the motor 1340, may be housed or coated with inert and/or sterile materials. The mixer 1300 may be coupled to the motor 1340 and/or the cap 1306 such that the mixer 1300 is suspended as described herein. The motor 1340 may be controlled by an analogue switch external of the vessel 1310 and/or cap 1306, an analogue switch within the cap 1306 that may be operated via exerting a motion or force on the cap 1306, e.g., tapping, screwing, twisting, pressing, etc., a remote/wireless controller, NFD activation, or the like.

[0079] Referring to FIGS. 14A, 14B, and 14C, a system for mixing a sample is illustrated including a mixer 1410 within a vessel 1407. The mixer 1410 is rotatably coupled to a scaffold 1403 such that the mixer 1410 is free to rotate with respect to the scaffold 1403 and the vessel 1407. The scaffold 1403 includes a lip 1411 having a diameter wider than a diameter of a top 1409 of the vessel 1407. The mixer 1410 includes a shaft 1405 having a longitudinal axis L and radial extension members 1406 extending radially from the longitudinal axis L about the shaft 1405. A mixer magnet 1401 is coupled to an end of the shaft 1405. The mixer 1410 is suspended away from a bottom surface of the vessel 1407 such that the radial extension members 1406 are free to rotate without contacting walls of the vessel 1407. Although four radial extension members 1406 are illustrated, in various embodiments any number of radial extension members may be employed, e.g., 1, 2, 3, 5, 6, 10, etc. A first O-ring 1402 is disposed about the shaft 1405 and between the scaffold 1403 and the mixer magnet 1401 (i.e., below the magnet). The first O- ring 1402 may suspend the mixer 1410 substantially out of contact with the scaffold 1403 to promote the mixer magnet 1401 to interact with another magnet (e.g., a magnet of a drive shaft) for rotation. In various embodiments, a second O-ring that may be substantially similar to the first O-ring 1402, can also be disposed between the scaffold 1403 and the shaft 1405 (i.e., below the scaffold 1403 and above the radial extension members 1406). This second O-ring 1402 may reduce or prevent frictional contact between the shaft 1405 with the scaffold 1403. A cap (not illustrated) may be configured to couple to the top 1409 of the vessel 1407.

[0080] Referring to FIGS. 15A and 15B, a system for mixing a sample is illustrated including a mixer 1510 rotatably coupled to a scaffold 1503. The scaffold 1503 is configured to couple to a vessel such that the mixer 1510 extends within a reservoir of the vessel without contacting walls of the vessel, as described herein. The mixer 1510 includes a shaft 1505 having a longitudinal axis L and a radial extension member 1506 extending radially from the longitudinal axis L about the shaft 1505. Although one radial extension member 1506 is illustrated, in various embodiments any number of radial extension members may be employed, e.g., 2, 3, 4, 5, 6, 10, etc. A mixer magnet 1501 is coupled to an end of the shaft 1505. The scaffold 1503 includes an aperture 1508 through the scaffold 1503 configured for external access into a vessel when the scaffold 1503 is coupled to the vessel. A ball-bearing 1502 is disposed between the shaft 1505 and the scaffold 1503 such that the shaft 1505 may rotate without contacting the scaffold 1503. A vessel (not illustrated) may be coupled to the scaffold 1503 and a top of the vessel may be coupled to a cap (not illustrated).

[0081] Referring to FIGS. 16A and 16B, a system for mixing a sample is illustrated including a mixer 1610 within a vessel 1607. The mixer 1610 is rotatably coupled to a scaffold 1603 such that the mixer 1610 is free to rotate with respect to the scaffold 1603 and the vessel 1607. The mixer 1610 includes a shaft 1605 having a longitudinal axis L and radial extension members 1606 extending radially from the longitudinal axis L about the shaft 1605. Although four radial extension members 1606 are illustrated, in various embodiments any number of radial extension members may be employed, e.g., 1, 2, 3, 5, 6, 10, etc. A mixer magnet 1604 is coupled to an end of the shaft 1605. The mixer 1610 is suspended away from a bottom surface of the vessel 1607 such that the radial extension members 1606 are free to rotate without contacting walls of the vessel 1610. The scaffold 1603 includes an aperture 1608 through the scaffold 1603 configured for external access into the vessel 1607 through the scaffold 1603. A ball-bearing 1602 is disposed between the shaft 1605 and the scaffold 1603 such that the shaft 1605 may rotate without contacting the scaffold 1603. A top 1609 of the vessel 1607 may be coupled to a cap (not illustrated) with the scaffold 1503 and mixer 1610 coupled within the vessel 1607.

Methods

A. General Methods

[0082] The present disclosure further provides methods of mixing a sample for calorimetry which comprises transferring or collecting a sample into a sealed vessel; suspending a mixer within the vessel such that the mixer is contactless with at least the bottom of the vessel; rotating the mixer; and measuring a metric of the sample. In some embodiments, suspending the mixer further comprises placing the vessel in axial alignment with a drive magnet, and rotating the mixer further comprises rotating the drive magnet. In some embodiments, rotating the mixer further comprises a magnetic coupling of a drive shaft magnet with a mixer magnet.

[0083] In some embodiments, the mixer further comprises a scaffold rotatably coupled to the mixer, the scaffold extending about a mixing portion of the mixer. In some embodiments, rotating the mixer further comprises a contactless magnetic coupling of a drive shaft magnet with a mixer magnet. In some embodiments, the mixer is directly coupled to a motor which is powered by a battery that resides within the sample vessel.

[0084] In accordance with the foregoing, the vessel can be disposed within a calorimeter and the measured metric is the heat flow generated by the sample.

[0085] Any of the methods above may be used in conjunction with to aseptically transferring or collecting blood directly into a sealed, sterilized vessel as may be done, for example, in a clinical setting. When blood is collected into a vessel with media and resins adapted for microbial growth in the presence of whole blood, the sample is a blood culture sample. Conveniently, blood transfer is accomplished by know blood collection methods into such vessels which has a hermetically sealed cap with a septum as part of the cap. Once collected and transferred to the clinical laboratory for interrogation, the vessel with the blood culture sample is placed in the calorimeter to allow growth of any blood borne microorganisms. In preferred embodiments calorimetric detection is by isothermal calorimetry to measure the heat flow associated with the blood culture sample.

Methods of Microbial Growth

[0086] In further embodiments of the disclosure, the devices and systems described herein are used to detect the presence or absence of microbial growth in a blood culture sample. The method comprises (a) incubating a blood culture sample under isothermal conditions for a time and at a temperature sufficient for microbial growth to occur. Such samples can be obtained by aseptic transfer, or collection, of blood into a sterile mixing device of the disclosure, i.e., a container or vessel, which contains (i) media adapted for growing microorganisms in the presence of whole blood, and (ii) a magnetic mixer as embodied herein; (b) mixing the sample by magnetically engaging the magnetic mixer into a position within the device to provide for mixing without the mixer contacting at least the bottom of said container; and (c) monitoring the heat flow produced by the blood culture sample to determine whether microbial growth is occurring.

[0087] In some embodiments, the device is hermetically sealed. In some embodiments, the magnetic mixer is can be coupled to a motor which is powered by a battery that resides within the mixing device.

[0088] The present methods are conducted under isothermal conditions suitable for detecting heat flow by continuous monitoring using calorimetry, and preferably isothermal calorimetry. Various forms of calorimetry are known to the skilled artisan for use as described herein.

[0089] In an embodiment, heat flow is monitored by isothermal calorimetry and such monitoring can be continuous or intermittent to detect microbial growth in a sample. Once growth is detected the sample can be directly or indirectly subjected to further analyses such as AST, phenotyping of the microorganism, or other diagnostic test to aid in clinical diagnosis and treatment decisions. Monitoring of the sample is continued until microbial growth is detected or until sufficient time has elapsed that the probability of obtaining a positive growth result is unlikely. Typically, the maximum period for monitoring is about 5 days but the period can also be based on the expected TTD for any particular microorganism under consideration or suspected to be in the blood culture sample. Hence, the useful monitoring period depends on many factors, including but not limited to, the culture medium, incubation temperature, degree of mixing and the microorganisms suspected. It is well within the skill of those in the art to determine the appropriate duration for the monitoring period.

[0090] In an embodiment, monitoring is continuous until microbial growth is detected, meaning that the sample remains in the calorimeter detection path and the heat flow is constantly measured. In an embodiment, monitoring is intermittent until microbial growth is detected, meaning that the sample can remain in the calorimeter detection path but heat flow is measured intermittently or meaning that the sample can be moved out of the detection path and then returned at predetermined time (or time intervals) into the detection path for heat flow detection. One advantage of intermittent monitoring is that it allows one to rotate monitoring among multiple samples by sharing the actual monitoring time. Samples waiting for monitoring can be held in the calorimeter box or removed to another incubation site. Further, if microbial growth is not detected from the sample after a predetermined time such as 5 days, the sample can be discarded or released from the system.

[0091] Conveniently, these methods can be used in automated platform systems which allow processing and monitoring of many samples using automated/robotic systems.

Sample collection

[0092] Sample collection and processing, including media suitable for growing microorganisms in the presence of whole blood, can be conducted or formulated as described hereinbelow. For example, one or more samples of whole blood are collected from a patient using known, standard procedures. Incubation of samples prior to interrogation is an important advancement as the healthcare system continues to migrate toward hub and spoke models where samples are transported to a central diagnostic laboratory that may be hours distance away. This transport can delay incubation and thus microbial sample detection by eight hours or more. The use of calorimetry and optional incubation proceeding calorimetry, as discussed in the present disclosure, decreases the TTD.

[0093] In embodiments, each sample comprises up to 10 mL of whole blood. In embodiments, 10 mL samples are used for subsequent processing as described herein. In other embodiments,

10 mL samples are divided into two or more subsamples or smaller volumes may be collected into two or more vessels. These subsamples may enable greater flexibility in sample processing and allow smaller volumes of sample per container. Alternatively, or additionally, the subsamples may be processed into separate workflows as described throughout the specification. [0094] In embodiments, the volume of whole blood collected may be measured by any suitable method including but not limited to volume, weight or heat capacity. Information regarding the volume of whole blood within a collection vessel may be incorporated into criteria for designation of sample containing microorganism.

[0095] In certain embodiments of the present disclosure, whole blood is collected into a consumable collection vessel comprising additives such as one or more anticoagulants; antimicrobial-adsorbing resins; and nutrient media. Additionally, one or more anti-foaming materials, such as polypropylene glycol, may be included. The consumable may be designed to specifically draw the blood sample in and may further have a pre-determined gas ratio present, as known to those skilled in the art to enhance microorganism growth.

[0096] Examples of anticoagulants that may be used in embodiments include sodium fluoride potassium oxalate, potassium oxalate, citrate-dextrose, sodium citrate, potassium ethylene- diamine-tetra- acetic acid (K2 EDTA), lithium heparin, sodium heparin, ammonium heparin and sodium polyanethole sulfonate (SPS).

[0097] Examples of antimicrobial-adsorbing resins that may be used in embodiments include AG MP-50 Resin, Amberjet 4200, Amberjet 4200 Na-i- form, Amberlite HPR100, Amberlite IR120, Amberlite IR120 H+ form, Amberlite IR120 H+ strongly acidic form, Amberlite IR120 Na+, Amberlite IRA402, Amberlite IRA402 Na-i- form, Amberlite XAD16N, Amberlite XAD-2, Amberlite XAD4, Amberlite XAD7HP, Resyn 101 H+ form, Resyn 101 Na-i- form Amberlyst 15, Amberlyst 15 H+ form, Bio-Beads SM-2, Dowex Marathon A, Dowex Marathon A Na-i- form, Dowex Marathon A H+ form, Amberlite HPR4800 OH, Lewatit C267, Lewatit MonoPlus SP 112 Na+, Lewatit VP OC 1065, Poly(styrene-co-divinylbenzene), Poly(styrene-co- divinylbenzene) microspheres 6-10 um, Poly(styrene-co-divinylbenzene) 200-400 mesh particle size 2% cross-linked, Cation membrane sheet, Nafion 212, Celgard 2400, and others as disclosed in US 4,174,277 and US 5,624,814, which are fully incorporated by reference herein. Some or all of the antimicrobial-adsorbing resins may be isolated from the anticoagulant and/or the nutrient media prior to the addition of blood sample. Additionally, resins may be supported on solid substrates, membranes or may be capable of magnetic capture. In some embodiments, resins remain within the sample during downstream interrogation.

[0098] Examples of nutrient media that may be used in embodiments include modified tryptic soy, brain heart infusion, modified Middlebrook broth or agar. Nutrient medias may be additionally supplemented to further support growth of microorganisms using components known to those skilled in the art.

[0099] In certain embodiments, the total volume of blood to equal one standard of care sample is a 10 ml whole blood sample. As described herein, a single 10 ml sample may be optionally collected into separate vessels, or subsequently split into separate fractions. Collection of the sample separate vessels would enable smaller volumes to be tested in the calorimeter. This may be advantageous as current large volume calorimeters are less sensitive than smaller volume calorimeters, larger volumes will require a longer time for the temperature to reach the desired incubation temperature, and longer time constants or calorimeter constant.

[0100] In certain embodiments, the ratio of blood to growth media and other additives may vary from 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10. Without wishing to be bound by theory, detection by calorimetry is less influenced by the microorganism concentration as compared to detection methods, such as optical. Thus, dilution of blood into growth media is not expected to dramatically reduce detection sensitivity given that the specifications of the detection instrument to do not change.

[0101] In an embodiment, following the introduction of blood into the consumable, incubation under conditions promoting microorganism growth may be performed. Viewed in the context of their potential for rapid culture determinations, the calorimetry-based methods of this disclosure are uniquely complemented by the incubation methods described herein. The current clinical practice of holding collected blood samples at room temperature prior to laboratory processing and measurement is predicated, in part, on the desirability of achieving log-phase growth during continuous monitoring processes, in order to differentiate the microbial signal from the mammalian cell baseline. Holding samples at reduced temperatures lowers the rate of microbial growth, thus reducing the risk that microbes will reach stationary phase before they enter the blood culture system. By contrast, in the calorimetry methods described herein, it may be valuable to maximize the potential thermal signal of microbes by maximizing the number of microbes present in the sample when it is monitored for heat flow. Thus, incubation under conditions that favor microbial growth ( e.g ., subjecting the sample to temperatures above room temperature, such as within the range 31-39°C, and/or mixing) may increase both the magnitude and rate of growth of positive heat flows by the time the sample is placed in the calorimeter.

This, in turn, may reduce the time required to achieve a positive result and/or reduce the risk of false negative results. Additionally, initial incubation prior to detection will further limit the time a sample dwells within the calorimeter, enabling higher sample throughput.

[0102] In a related embodiment, incubation of blood samples prior to interrogation can occur immediately after the sample is collected, upon entry of the sample into a transit system or at any time following sample collection. This incubation may optionally occur in a portable device.

The portable device may register the time at which a sample is added to the system. The device may optionally contain a non-calorimetry method for detection of microbial growth. To account for potential differences in transit or incubation time, incubation temperatures may be raised or lowered at any time during the incubation, or incubation may be stopped or paused and re-started at any time before interrogation.

[0103] Mixing may be performed prior to and/or during incubation and interrogation.

Interrogation of Samples for Microbial Growth by Calorimetry Analysis [0104] Embodiments described herein utilize heat flow analysis for the rapid detection of microbial growth in whole blood samples. In preferred embodiments, heat flow from samples is monitored using calorimetry, such as adiabatic calorimetry, semiadiabatic calorimetry, drop heat capacity calorimetry, vaporization-sorption calorimetry, reaction calorimetry, constant pressure calorimetery, isothermal calorimetry or differential scanning calorimetry. Isothermal calorimetry is generally preferred over calorimetry methods, but other forms of calorimetry may be used in the methods described herein. Systems enabling multiple samples to be run in parallel are preferred, such as the TA Instruments (Wakefield, MA) model TAM IV-48, model TAM III, model Air 8-channel and 6-channel, the Omnical (Stafford, TX) Insight, Thermal Hazard Technology (THT; Bletchley, UK) model pMC or the Symcel (Solna, Sweden) calScreener. [0105] It should be appreciated that other calorimetry detection equipment having the requisite sensitivity to heat flow from whole blood samples may be used in conjunction with the methods described herein.

[0106] The presence of microbial growth within a whole blood sample may be determined based on absolute and/or relative heat flow from the sample under test. Typically, heat flow from blood samples will decrease over time, for example due to a decrease in metabolic heat production over time. In accordance with embodiments described herein, the inventors have found, under certain conditions, the expected decrease in heat flow from whole blood samples is decreased by microbial growth. In other words, under certain conditions, the decrease in expected heat flow is slowed by microbial growth such that the rate of decrease in heat flow is decreased, eliminated, or reversed for some period of time.

[0107] Samples may be loaded into the calorimeter on a random-access basis or may be batched for loading at discrete time intervals. Such time intervals may be every 1, 2, 3, 4, 5, 6, 7, or 8 hours. In an embodiment, samples in the calorimeter may be incubated isothermally under conditions promoting microorganism growth, such as between 31-39°C. In alternative embodiments, differential scanning calorimetry may be used within a suitable temperature range, such as 20-40°C. In alternative embodiments the samples introduced to the calorimeter may have a nutrient agar present, such that solid-phase growth may occur. In alternative embodiments the samples introduced to the calorimeter may have nutrient agar present as well as nutrient broth, such that microorganisms may be grown in both solid- or liquid-phases.

[0108] Certain embodiments of this disclosure also incorporate a staged analysis workflow in which samples under test for microorganism growth in the calorimeter may reside within the calorimeter for a predetermined period of time, such as 12, 24, 48, 60, 72 or more hours.

Samples that do not test positive during this time period may be transferred to one or more additional interrogation processes, such as optical, pH, gaseous or electrical methods. In this way, space in the calorimeter may be conserved and utilized for rapid results, whereas slower- growing samples may be measured using a lower-cost method. In other embodiments, samples removed from the calorimeter are reintroduced into the calorimeter after a predetermined period of time removed from the calorimeter. Such samples may be incubated at temperatures greater than 20°C before reintroduction into the calorimeter. [0109] Additionally, the inventors have found that, in some cases, signals from duplicate subsamples taken from the same sample can be added together to accurately identity the presence of microbial growth. Thus, certain methods of this disclosure comprise splitting one or more samples into subsamples, or obtaining and interrogating multiple samples from the same patient, and adding the signals measured therefrom to assess microbial growth. This can be done, e.g., if growth is not detected in either sample or subsample from a patient, prior to removing the sample(s) from the calorimeter and monitoring them by the secondary detection method. Optionally, if microbial growth is detected from one sample fraction, the other fractions from the same sample may also considered positive and later combined or handled using different subsequent methods.

[0110] In an embodiment, samples for which microorganism growth is detected may be removed from the calorimeter directly following positive growth determination or may continue incubating in the calorimeter for a defined period of time for sample batching purposes. Positive growth determinations may be based on absolute or relative heat flows.

[0111] Further sample analysis and post-interrogation processing may be conducted as described in provisional patent application entitled “Rapid Methods for Determining Microorganism Growth in Whole Blood Samples,” filed on even date herewith.

[0112] While some embodiments have been described by way of illustration, it will be apparent that the embodiments can be put into practice with many modifications, variations and adaptations, and with the use of numerous equivalents or alternative solutions that are within the scope of persons skilled in the art, without departing from the spirit of the invention or exceeding the scope of the claims.

[0113] All publications, patents, and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

EXAMPLES

[0114] The examples presented herein represent certain embodiments of the present disclosure. However, it is to be understood that these examples are for illustration purposes only and do not intend, nor should any be construed, to be wholly definitive as to conditions and scope of this invention. The examples were carried out using standard techniques, which are well known and routine to those of skill in the art, except where otherwise described in detail.

Example 1. Sample Mixing Reduces Time to Detect Microbial Growth

[0115] To prepare samples for analysis, negative human blood was drawn into SPS tubes and 10 mL of this SPS-treated blood was added to a Bactec plus media bottle and inverted to mix. After mixing, 18 mL was withdrawn from the bottle and added to a calorimetry vessel containing 1 mL of anti-microbial adsorbing resins (18 mL/vessel) and inoculated with 100 CFU of K. pneumoniae. Samples were placed into a TAMAir calorimeter and incubated at 37°C with mixing using the TAMAir Admixer or without mixing. The heat flow of the samples was monitored, and the TTD was recorded as the time when the increase in heat was > 10 pW. The TTD for the mixed sample was 5.85 hours (Fig. 1, line A), whereas the TTD for the non-mixed sample was 7.1 hours (Fig. 1, line B). In comparison to one of the current standards of care, the TTD for a Bactec plus bottle containing the same donor blood, inoculum and resins was 10.33 hours measured in the Bactec 9000 series system. Additionally, the maximum heat flow observed for the mixed sample was >6 mW, whereas the maximum heat flow for the non-mix sample was approximately 1.5 mW.

Example 2. Effect of Sample Mixing on Calorimetric Detection of Heat Flow

[0116] Samples were prepared and transferred to calorimetry vessels as described in Example 1 but without adding bacteria or resins. Fig. 2 shows the heat flow over time as measured in a TAMAir calorimeter for (A) a sample mixed with the TAMAir Admixer on the highest mixing speed and (B) a non-mixed sample. Over the time period shown (~an hour), the mixed sample varied by ~4 pW heat flow while the non-mixed sample had a negative slope and decreased by -24 pW.

Example 3. Sample Mixing Reduces Time to Detect Candida sps. Growth

[0117] Negative SPS-treated human whole blood was inoculated with either C. auris or C. tropicalis at 100 CFU/mL (non-mixing experiment) or 30 CFU/mL (mixing experiment). To prepare samples for growth and analysis, 10 mL of the inoculated blood was injected into a Bactec Plus Aerobic medium bottle and incubated for 3 hours in a Bactec 9050. After incubation, 18 mL was withdrawn and added to a calorimetry vessel (ampoule) without added resins. The ampoules had either a crimp top for the non-mixing experiments or the admixer attached to the ampoule for the mixing experiments. The sample and reference ampoules were incubated at 37°C in a heat block for 30 minutes before transfer to the TAMAir calorimeter. The TTD of the samples was recorded as the time point at which the heat flow exceeded 10 pW. Fig. 3 shows the TTD for each species for mixed (solid bars) and non-mixed samples (open bars). These mixing effects are also provided in Table 1 which provides a calculation of the percent improvement in TTD for mixed versus unmixed samples. The reported TTDs include the 3-hour pre-incubation time in the Bactec 9050 for the data shown in Fig. 3 and in Table 1.

TABLE 1. Change of TTD with Mixing for Candida sps.

Example 4. Effect of Sample Mixing on Various Species

[0118] Samples were prepared and transferred to calorimetry vessels as described in Example 1 using inoculums of from 5-100 CFU of the bacterial species indicate in Table 2. For each species, one sample was mixed using a traditional overhead mixer and other sample was not mixed. The TTD of the calorimetry samples was recorded when the heat flow exceeded 10 pW and TTDs were compared for mixed and non-mixed samples as shown in Table 2. In all cases, the TTD was reduced with mixing, with the percent improvement provided Table 2. TABLE 2. Change of TTD with Mixing for Various Microorganisms

Example 5. Mixer Design Limits Friction

[0119] Tape was added to the mixer foot of a TAMAir Admixer, causing it to drag across the interior surface of the sample vessel during mixing (Fig. 9A). Heat flow from the vessel was monitored with mixer off, at low speed (30 rpm), with mixer off and at high speed (120 rpm) (Fig. 9B). Mixing on the slowest setting resulted in heat flow fluctuations up to 0.7 mW, whereas heat flow fluctuations of the sample when the mixer was present, but stationary was typically <1 pW. With mixing at the fastest setting, large heat flow fluctuations of up to 8.6 mW were observed.

Example 6. Effects of Mixing versus Non-Mixing

[0120] Samples were prepared, inoculated with S. aureus , and transferred to calorimetry vessels as described in Example 1. The samples and a non-inoculated, sterile control sample were placed in a calorimeter at 37°C. The heat flow of the samples was monitored, and TTD recorded as the time at which the increase in heat flow was > 10 pW. One sample was mixed with a magnetic mixer of the disclosure (Fig. 10, line A); the second sample (Fig. 10, line B) and the control sample were unmixed (Fig. 10, line C).

Example 7. Comparison of a Magnetic Mixer and an Overhead Internal Mixer

[0121] Samples were prepared, inoculated with K pneumoniae , and transferred to calorimetry vessels as described in Example 1. The samples were placed in a calorimeter at 37°C. The heat flow of the samples was monitored, and TTD recorded when the increase in heat flow was >

10 pW. Sample A was mixed with a magnetic mixer of the disclosure (Fig. 11, line A); Sample B was mixed with a TAMAir Admixer (Fig. 11, line B).

Claims

WE CLAIM:

1. A system for mixing a sample, comprising: a mixer comprising a first end, a second end, and a longitudinal axis therethrough; a mixer magnet coupled to the mixer along the longitudinal axis; a sealed vessel containing the mixer; a drive shaft spaced a distance away from the mixer, the drive shaft extending substantially axially along the longitudinal axis and rotatable about the longitudinal axis; and a drive magnet coupled to the drive shaft.

2. The system of claim 1, wherein the mixer is magnetically suspended such that the mixer is contactless with at least a bottom surface of the vessel.

3. The system of claim 1, wherein the drive magnet is coupled to the drive shaft via a radial member extending radially away from the drive shaft.

4. The system of claim 1, further comprising a scaffold rotatably coupled to the mixer, the scaffold extending about a mixing portion of the mixer.

5. The system of claim 1, wherein the mixer comprises at least one radial member extending radially away from the longitudinal axis.

6. The system of claim 1, wherein at least one of the mixer magnet and the drive magnet comprise a diametric magnet.

7. The system of claim 1, wherein the mixer comprises one of a rod, a radial extension, a projection, an impeller, a propeller, a turbine, a fan, a bar, a paddle, a vane, and a helix.

8. The system of claim 1, further comprising a scaffold coupled to a top portion of the vessel and wherein the mixer is rotatably coupled to the scaffold.

9. A mixing device for mixing a sample comprising: a vessel comprising a reservoir; a magnetic mixer disposed within the reservoir comprising: a mixer comprising a first end, a second end, and a longitudinal axis therethrough; a magnet coupled to the mixer along the longitudinal axis; and a cap coupled to the vessel.

10. The mixing device of claim 9, further comprising a scaffold rotatably coupled to the mixer, the scaffold extending about a mixing portion of the mixer.

11. The mixing device of claim 9, wherein the magnet comprises a diametric magnet.

12. The mixing device of claim 9, wherein the mixer comprises at least one radial member extending radially away from the longitudinal axis.

13. The mixing device of claim 9, further comprising a scaffold coupled to a top portion of the vessel and wherein the mixer is rotatably coupled to the scaffold.

14. The mixing device of claim 9, wherein the mixer comprises one of a rod, a radial extension, a projection, an impeller, a propeller, a turbine, a fan, and a helix.

15. The mixing device of any one of claims 9-14, wherein said sample is a blood culture sample.

16. The mixing device of any one of claims 9-15, wherein said cap comprises a septum.

17. The mixing device of any one of claims 9-16, wherein said cap is hermetically sealed to the vessel.

18. The mixing device of any one of claims 9-17, wherein said vessel further contains media adapted for growing microorganisms in the presence of whole blood.

19. The mixing device of any one of claims 9-18, wherein said mixing device is sterilized.

20. The mixing device of any one of claims 9-19, wherein said vessel is adapted for isothermal calorimetry.

21. A method of mixing a sample, comprising: transferring or collecting a sample into a sealed vessel; suspending a mixer within the sealed vessel such that the mixer is contactless with at least the bottom of the sealed vessel and with a drive shaft configured for rotating the mixer; rotating the mixer; and measuring a metric of the sample.

22. The method of claim 21, wherein suspending the mixer further comprises placing the vessel in axial alignment with a drive magnet, and rotating the mixer further comprises rotating the drive magnet.

23. The method of claim 21, wherein a scaffold is rotatably coupled to the mixer, the scaffold extending about a mixing portion of the mixer.

24. The method of claim 21, wherein measuring a metric of the fluid sample further comprises disposing the vessel within a calorimeter.

25. The method of claim 24, further comprising suspending the vessel within the calorimeter such that the vessel is contactless with a surface of the calorimeter.

26. The method of claim 21, wherein rotating the mixer further comprises a contactless magnetic coupling of a drive shaft magnet with a mixer magnet.

27. The method of any one of claims 21-26, wherein transferring or collecting is aseptically transferring or aseptically collecting into sealed, sterilized vessel

28. The method of any one of claims 21-27, wherein said sample is a blood culture sample.

29. The method of any one of claims 21-28, wherein aseptic transfer is accomplished through a septum forming part of the seal of said vessel.

30. The method of any one of claims 21-29, wherein said seal is a hermetic seal.

31. The method of any one of claims 21-30, wherein said vessel further contains media adapted for growing microorganisms in the presence of whole blood.

32. The method of any one of claims 21-31, wherein said vessel is adapted for isothermal calorimetry.

33. The method of any one of claims 21-32, wherein a scaffold is coupled to a top portion of the vessel and wherein the mixer is rotatably coupled to the scaffold.

34. A method of detecting microbial growth in a blood culture sample which comprises (a) incubating a blood culture sample under isothermal conditions for a time and at a temperature sufficient for microbial growth to occur, wherein said blood culture sample is obtained by aseptic transfer of blood into the reservoir of a sterile mixing device of any one of claims 9-20, which reservoir has a content comprising (i) media adapted for growing microorganisms in the presence of whole blood, and (ii) a magnetic mixer;

(b) mixing said sample by magnetically engaging the magnetic mixer into a position within said mixing device that provides for mixing without said mixer contacting at least the bottom of the mixing device; and

(c) monitoring the heat flow produced by said sample to determine whether microbial growth is occurring.

35. The method of claim 34, wherein said device is hermetically sealed.

36. The method of claim 34 or 35, wherein the magnetic mixer is engaged with the top of the vessel, the lid or a scaffold coupled with the vessel.

37. The method of any one of claims 34-36, wherein mixing is battery powered.

38. The method of claim 37, wherein battery powered mixing is activated by a wireless signal or mechanically.

39. The method of any one of claims 34-38, wherein the isothermal conditions are suitable for detecting heat flow by continuous monitoring or intermittent monitoring using isothermal calorimetry

40. The method of any one of claims 34-39, wherein heat flow is monitored by isothermal calorimetry.

41. The method of any one of claims 33-40, wherein monitoring is continuous or intermittent until microbial growth detected

42. The method of any one of claims 34-41, wherein said mixing device is designed for use in an automated platform system.

43. A system for mixing a sample, comprising: a mixer comprising a first end, a second end, and a longitudinal axis therethrough; a motor coupled to the mixer along the longitudinal axis; a battery powering the motor; and a vessel containing the mixer, the motor, and the battery.

44. The system of claim 43, wherein the vessel is configured to incubate a blood culture sample under isothermal conditions for a time and at a temperature sufficient for microbial growth to occur, wherein the blood culture sample is obtained by aseptic transfer of blood, and wherein the vessel contains a media adapted for growing microorganisms in the presence of whole blood.

45. The system of claim 43, wherein the vessel is hermetically sealed.

46. The system of claim 43, wherein the mixer is configured to engage at least one of a top of the vessel and a scaffold coupled to the vessel.

47. The system of claim 43, wherein the motor is configured to activate by a wireless signal or a mechanical switch.

48. The system of claim 43, wherein the vessel is configured to incubate a blood culture sample under isothermal conditions suitable for detecting heat flow by monitoring via isothermal calorimetry.

49. The system of claim 43, wherein the vessel is configured to incubate a blood culture sample under isothermal conditions suitable for detecting microbial growth.

50. The system of claim 43, wherein the mixer is configured for use in an automated platform system.