WO2021046504A1

WO2021046504A1 - Infection detection systems and methods including a sample processor having integrated sample filter and meter

Info

Publication number: WO2021046504A1
Application number: PCT/US2020/049642
Authority: WO
Inventors: John C. VICTOR; Kristine Marie SIM; Kweku Amissah ADDAE-MENSAH; Rodney Wilmer DENLINGER; Victoria E. WAGNER; Robert Ulrich
Original assignee: Teleflex Medical Incorporated
Priority date: 2019-09-06
Filing date: 2020-09-08
Publication date: 2021-03-11

Abstract

An infection detection system includes a sampling device, a lysing chamber, a filter, a meter, a NASBA fluidic network, and an instrument. The sampling device is configured to contain a whole blood sample containing a pathogen target. The lysing chamber is configured to be in fluid communication with the sampling device to receive the whole blood sample. The lysing chamber is configured to lyse the whole blood sample into a lysate. The filter is configured to be in fluid communication with the lysing chamber and to filter the lysate into a filter lysate. The meter is configured to be in fluid communication with the filter and configured to meter a predetermined amount of filtered lysate from the filtered lysate. The NASBA fluidic network is configured to be in fluid communication with the meter to receive the predetermined amount of filtered lysate.

Description

INFECTION DETECTION SYSTEMS AND METHODS INCLUDING A SAMPLE PROCESSOR HAVING INTEGRATED SAMPLE FILTER AND METER

Cross-Reference to Related Application

[0001] This application claims priority to U.S. Provisional Patent Application No. 62/897,178, filed September 6, 2019, the disclosure of which is incorporated by reference herein in its entirety.

Technical Field

[0002] This application relates generally to systems, devices, and methods for collecting a sample and for processing the sample in an infection detection system.

Background

[0003] Patient exposure to health care environments elevates risk of systemic and/or local infections caused by commensal microorganisms. Infection risk is particularly elevated during the use of external communicating medical devices. There is accordingly a need for systems, devices, and methods for rapidly collecting, identifying, quantifying, and characterizing causative microorganisms associated with infections.

[0004] For any Point-of-care diagnostic system, the ability to collect the required amount of sample and transfer that sample to the testing system is critical. Existing methods that are used to collect biological samples for laboratory testing may collect too much sample as these methods are generally tailored to suit laboratory testing equipment utilized in central testing facilities. For example, venous blood collected for culture and testing can be as much as 20ml of blood. Such a large sample is not amenable for a Point-of-care testing system. There is therefore the need to take these large sample volumes/sizes collected using conventional sample collection methods and reduce them to appropriate sizes without any appreciable loss in biological signal or target of interest.

Summary

[0005] The present inventors recognize that there is a need to improve one or more features of infection detection systems and methods. This invention proposes various methods to transfer sample in liquid form from a syringe or vacuum collection tube to a microfluidic cartridge. In some embodiments of the invention, the transfer mechanism simultaneously meters the transferred sample to a specific volume during the transfer to the microfluidic system. These embodiments simplify the steps of use for the clinicians by eliminating the need for manual laboratory equipment for dilution methods and centrifuging of the samples.

[0006] Aspects of the present invention include an infection detection system having a sampling device configured to contain a whole blood sample containing a pathogen target, a lysing chamber configured to be in fluid communication with the sampling device to receive the whole blood sample, the lysing chamber being configured to lyse the whole blood sample into a lysate, a filter configured to be in fluid communication with the lysing chamber and to filter the lysate into a filter lysate, a meter configured to be in fluid communication with the filter and configured to meter a predetermined amount of filtered lysate from the filtered lysate, and a NASBA fluidic network configured to be in fluid communication with the meter to receive the predetermined amount of filtered lysate. The NASBA fluidic network including an enzyme and a primer for amplifying a predetermined genetic sequence in the pathogen target contained within the predetermined amount of filtered lysate, and a beacon that is configured to attach to the predetermined genetic sequence in the pathogen target. The infection detection system further comprising an instrument configured to identify the beacon when the beacon is attached to the pathogen target to signal a presence of the pathogen target.

[0007] Further aspects of the present invention include a method of detecting an infection using the infection detection system. The method inlcuidng collecting the whole blood sample containing the pathogen target, lysing the whole blood sample into the lysate, filtering the lysate into the filtered lysate, metering the predetermined amount of filtered lysate, amplifying the predetermined genetic sequence in the pathogen target contained within the predetermined amount of filtered lysate, attaching the beacon to the pathogen target, and exciting the beacon attached to the pathogen target to signal the presence of the pathogen target.

[0008] Further aspects of the present invention include an infection detection system having a sampling device configured to contain a whole blood sample containing a pathogen target, and a sample processor. The sample processor including a meter configured to be in fluid communication with the sampling device and configured to meter a predetermined amount of the whole blood sample, a lysing chamber configured to be in fluid communication with the meter, the lysing chamber being configured to lyse the predetermined amount of the whole blood sample into a lysate, and a NASBA fluidic network configured to receive the lysate. The NASBA fluidic network having an enzyme and a primer for amplifying a predetermined genetic sequence in the pathogen target contained within the lysate, and a beacon that is configured to attach to the predetermined genetic sequence in the pathogen target. The infection detection system further including an instrument configured to identify the beacon when the beacon is attached to the pathogen target to signal a presence of the pathogen target.

[0009] The infection detection system further comprising a filter configured to be in fluid communication with the lysing chamber to filter the lysate prior to fluid communication of the lysate to the NASBA fluidic network.

[0010] The whole blood sample is contained within the sampling device at a pressure lower than a pressure the sample processor such that the whole blood sample is configured to be driven from the sampling device to the conduits within the sample processor when the sampling device is connected to the sample processor.

[0011] The sample processor includes an activator that is configured to activate fluid communication of the whole blood sample from the sampling device to the sample processor.

[0012] The activator is an air blister that is configured to be in fluid communication with the sampling device such that depression of the air blister is configured to impart a positive pressure within the sampling device to activate fluid communication of the whole blood sample from the sampling device to the sample processor.

[0013] The sample processor includes a sample tube port having a first cannula and a second cannula, the first cannula being in fluid communication with the air blister and the second cannula being in fluid communication with the meter.

[0014] The air blister is located adjacent to the sample tube port.

[0015] The air blister is located within the sample tube port and is configured to be depressed upon connection of the sampling device and the sample tube port.

[0016] The instrument and a cartridge interface comprise the activator, and the instrument is configured to impart a positive pressure, via the cartridge interface, within the sampling device to activate fluid communication of the whole blood sample from the sampling device to the sample processor. The sample processor includes a sample tube port having a first cannula and a second cannula, the first cannula being in fluid communication with the cartridge interface and the second cannula being in fluid communication with the meter.

[0017] The activator is a piston integrated into the sample processor and configured to selectively draw the whole blood sample from the sampling device.

[0018] The sample processor comprises a syringe port in fluid communication with the meter for connecting with a syringe and providing an alternative source of whole blood. [0019] The sample processor comprises a valve for controlling fluid flow within the sample processor.

[0020] The infection detection system further includes a blood press syringe having a plunger having a seal, a barrel including a cavity configured to receive the whole blood sample and to slidingly receive the plunger, and a filter comprising wire mesh attached to the barrel and configured to separate clear liquids from proteins and solids in a whole blood sample when the plunger forces the whole blood sample through the filter.

[0021] The primer includes any one or more of oligonucleotide sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO:

33, SEQ ID NO: 34, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 73, SEQ ID NO:

74, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 93, and SEQ ID NO: 94.

[0022] The beacon includes any one or more of oligonucleotide sequences SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 31, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 47, SEQ ID NO: 51, SEQ ID NO: 55, SEQ ID NO: 59, SEQ ID NO: 63, SEQ ID NO: 67, SEQ ID NO: 71, SEQ ID NO: 75, SEQ ID NO: 79, SEQ ID NO: 83, SEQ ID NO:

87, SEQ ID NO: 91, and SEQ ID NO: 95.

[0023] The lysing chamber comprises a lysis solution for lysing microorganisms in the whole blood sample, the lysis solution comprising 2 mM to 16 mM of a Quanidinium thiocyanate/guanidine thiocyanate, 20 mM to 160 pM of a Tris HCL, pH 8.5, 6 pM to 48 pM of a Magnesium chloride, 35 pM to 280 pM of a Potassium chloride, and 0.1% v/v to 1.0% v/v of an octylphenoxypolyethoxyethanol.

[0024] Further aspects of the invention include a method of detecting an infection using the infection detection system, the method including collecting the whole blood sample containing the pathogen target, metering the predetermined amount of the whole blood sample, lysing the metered whole blood sample into the lysate, filtering the lysate into the filtered lysate, amplifying the predetermined genetic sequence in the pathogen target contained within the predetermined amount of filtered lysate, attaching the beacon to the pathogen target, and exciting the beacon attached to the pathogen target to signal the presence of the pathogen target.

[0025] The lysing step is accomplished using the lysis solution comprising 2 mM to 16 mM of a Quanidinium thiocyanate/guanidine thiocyanate; 20 mM to 160 mM of a Tris HCL, pH 8.5; 6 pM to 48 pM of a Magnesium chloride; 35 pM to 280 pM of a Potassium chloride; and

0.1% v/v to 1.0% v/v of an octylphenoxypolyethoxyethanol.

[0026] There are, of course, additional aspects of the various embodiments of the invention disclosed herein that will be described below and which will form the subject matter of the claims. In this respect, before explaining at least one aspect of the invention in detail, it is to be understood that the invention is not limited in its application to the details of construction and to the arrangements of the components set forth in the following description or illustrated in the drawings. The invention is capable of aspects in addition to those described and of being practiced and carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein, as well as the Abstract, are for the purpose of description and should not be regarded as limiting.

[0027] As such, those skilled in the art will appreciate that the conception upon which this invention is based may readily be utilized as a basis for the designing of other structures, methods, and systems for carrying out the several purposes of the invention. It is important, therefore, that the claims be regarded as including such equivalent constructions insofar as they do not depart from the spirit and scope of the invention.

Brief Description of Drawings

[0028] In order that the invention may be readily understood, aspects of the invention are illustrated by way of examples in the accompanying drawings; however, the subject matter is not limited to the disclosed aspects.

[0029] FIG. 1 illustrates a schematic infection detection system in accordance with aspects of the invention.

[0030] FIGS. 2-4 illustrate views of aspects of an infection detection system in accordance with an embodiment of the invention having an air blister adjacent to a sample collection port.

[0031] FIGS. 5-7 illustrate views of aspects of an infection detection system in accordance with an embodiment of the invention having an air blister disposed within a sample collection port. [0032] FIGS. 8-10 illustrate views of an infection detection system in accordance with an embodiment of the invention having an instrument that activates displacement of the whole blood sample.

[0033] FIGS. 11 and 12 illustrate views of an infection detection system in accordance with an embodiment of the invention having a piston that that activates displacement of the whole blood sample.

[0034] FIG. 13 illustrates a blood press syringe in accordance with aspects of the invention.

[0035] Features of the infection detection system according to aspects of the invention are described with reference to the drawings, in which like reference numerals refer to like parts throughout.

Detailed Description

[0036] FIG. 1 shows an schematic representation of an exemplary infection detection system 10 in accordance with aspects of the invention. The infection detection system 10 is configured to process a sample and to determine whether the sample contains one or more predetermined pathogens. The infection detection system 10 in accordance with embodiments of the invention includes a sampling device 20, a lysing chamber 30, a filter 40, a meter 50, a nucleic acid sequence-based (NASBA) fluidic network 60, and an instrument 70. The infection detection system 10 also includes a sample processor 80, such as a cartridge, which at least includes the NASBA fluidic network 60 and may include any or all of the lysing chamber 30, the filter 40, and the meter 50. The sample processor 80 is configured to connect to the sampling device 20 and to receive and process a sample contained within the sampling device 20. The sample processor 80 may be disposable and replaceable, and may be adapted to process the collected sample using at least one NASBA assay. The infection detection system 10 may process the sample and determine whether the sample contains one or more predetermined pathogens rapidly, for example within an hour, thirty minutes, or less. The infection detection system 10 may process the sample and determine whether the sample contains one more predetermined pathogens at the point-of- care, for example within the same building, room, etc. as the patient. The infection detection system 10 thus eliminates the need for time-wasting intermediary treatment, storage, and/or extraneous transport of the sampling device 20. According to aspects of the invention, the entirety of the sample processing may occur within the various components of the infection detection system 10 thereby obviating the need of direct user intervention with the sample after the sample is collected. The infection detection system 10 may accordingly be used by a user of low skill and may be readily transported to and applied in a variety of environments (e.g., the home, a hospital room, etc.). As a result, infection in a patient may be rapidly detected and identified, which may improve the prognosis of the patient.

[0037] In accordance with a variety of embodiments of the present disclosure, pathogenic microorganisms and/or sequences related to antibiotic resistance are detected in a biological sample obtained from a patient. For the purposes of this disclosure, a biological sample includes whole blood, serum, plasma, cerebrospinal fluid (CSF), urine, synovial fluid, breast milk, sweat, tears, saliva, semen, feces, vaginal fluid or tissue, sputum, nasopharyngeal aspirate or swab, lacrimal fluid, mucous, or epithelial swab (buccal swab), and tissues (e.g., tissue homogenates), organs, bones, teeth, among others). For the purposes of this disclosure, a pathogenic microorganism includes, for example, one or more of Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus epidermidis, Candida parapsilosis , Streptococcus pneumoniae, Enterobacter cloacae complex, Haemophilus influenzae, Neisseria meningitidis, and Enterobacter aerogenes. For the purposes of this disclosure, an antibiotic resistance includes, for example, resistance to one or more of Fluconazole, Methicillin, Carbapenem, and Vancomycin. More particularly, the pathogenic organisms and/or antibiotic resistance markers may include those listed in Table I. More particularly still, the target sequences of the pathogenic organisms and/or antibiotic resistance genes may be detected using the various forward primers, reverse primers, and molecular beacons listed in Table I (which sets forth and defines SEQ. ID. Numbers 1 through 96).

Table I

[0038] The forward primers, reverse primers, and molecular beacons listed in Table I are particularly suitable for use in the infection detection system 10. In this regard, these forward primers, reverse primers, and molecular beacons are optimized for use with a NASBA amplification and detection system. For example, the primer and beacons described below in reference to the NASBA fluidic network 60 may include any of the primers and beacons listed in Table 1.

[0039] A lysing solution suitable for use in the infection detection system 10 quickly lyses microbial cell wall and membranes. In a particular example, the lysing solution may facilitate this lysis at room temperature and without physio-mechanical cell disruption.

In addition, the lysing solution may be benign to RNA and stable at room temperature. A specific example of a suitable lysing solution is found in Table II:

Table

[0040] It is an advantage of the lysing solution according to Table II that it is suitable for use in lysing a variety of gram positive, gram negative, and fungal microorganisms. In addition, it is an advantage of the lysing solution according to Table I that it has a viable shelf life of greater than 1 year of storage at room temperature. In addition, it is an advantage of the lysing solution according to Table I that it has a viable shelf life of greater than 1 year of storage at negative twenty degrees Celsius (-20°C). Of note, IGEPAL CA-630^® is a nonionic, non-denaturing detergent having the IUPAC name of octylphenoxypolyethoxyethanol. The lysing agent described below may correspond to any of the lysing solutions listed in Table II.

[0041] The sampling device 20 of the infection detection system 10 may be adapted to collect a sample, such as blood (e.g., whole blood), urine, fecal matter, purulence/pus, etc. Whole blood, as used herein, means blood drawn directly from a patient from which none of the components, such as plasma, platelets, or pathogens, has been removed. The sampling device 20 may collect the sample from a medical device (not shown). For example, the sampling device 20 may be exposed for a predetermined and/or extended period of time to an internal space or lumen in the medical device so as to collect a sample of any pathogen which may form in said space and/or lumen. The medical device may be an external communicating device used for treating a patient, such as a Foley catheter, a vascular catheter, a suction catheter, a bronchial scope, a urinary drain line, a respiratory suction catheter, a Bronco- Alveolar-Lavage Catheter, etc. The sampling device 20 may additionally or alternatively be adapted to collect a sample directly from a sample source such as urine, fecal matter, purulence/pus, a suspected infection site (such as a surgical dressing, wound, and/or an insertion site), etc. The sampling device 20 may additionally or alternatively be adapted to collect a sample intravenously, subcutaneously, or intraosseously. The sampling device 20 may be disposable and replaceable. According to aspects of the invention, the sampling device 20 may include a sample collection tube 400. The sample collection tube 400 may be a standard blood collection vacuum tube 400 containing a whole blood sample. Additionally or alternatively, the sampling device 20 may be a standard syringe 402 containing a whole blood sample. [0042] The lysing chamber may be any chamber configured to receive the whole blood sample and lyse the whole blood sample into a lysate. The lysing chamber may be in fluid communication with the sampling device 20. Fluid communication, as used herein, may mean that the structures in question are fluidly connected via any of a number of structures such as tubing, conduits, etc., that allow fluid to travel from one structure to another. In embodiments of the invention, flow of the whole blood sample from the sampling device 20 to the lysing chamber may be operatively connected to the instrument 70 and may be controlled and/or driven by the instrument 70. Throughout this disclosure, reference is made to the instrument 70 controlling and/or driving fluid flow, for example flow of the whole blood sample from the sampling device 20 to the lysing chamber. The instrument 70 may control and/or drive fluid flow when operatively connected to a fluid pathway and via any number of known fluid control systems, which may for example include pumps, valves, conduits etc. Further, the instrument 70 may control and/or drive fluid flow without physically contacting the fluid. Accordingly, the sample collector and/or the sample processor 80 maybe disposed and replaced while the instrument 70 may be used repeatedly without contaminating the samples.

[0043] The lysing chamber may include all of the materials for lysing the whole blood sample and pathogen cells contained therein and for extracting and purifying pathogen messenger RNA (i.e., dissolve targeted mRNA and remove inhibitors that could interfere with nucleic acid amplification). For example, the lysing chamber may include a lysing agent, such as a lyophilized Acris lysing chemistry, that is configured to lyse the whole blood sample into the lysate. Additionally or alternatively, the lysing chamber may physically lyse the whole blood sample ultrasonically or by freezing the whole blood sample.

[0044] According to one aspect of the invention shown in FIG. 1, the lysing chamber may include a plurality of chambers, for example, a first chamber and a second chamber, The first chamber may include a lysing chemistry, such as, the lyophilized Acris lysing chemistry. The lysing chemistry contained within the first chamber may be in the form of a reagent plug(s) having dried lysis reagents. The first chamber may be in fluid communication with the sampling device 20 and may receive the whole blood sample from the sampling device 20. In embodiments of the invention, the instrument 70 may control and/or drive flow of the whole blood sample from the sampling device 20 to the first chamber. Additionally or alternatively, the whole blood sample may be driven from the sampling device 20 to the first chamber via gravity, capillary flow, etc. The second chamber may include a diluent and may be in fluid communication with the first chamber. The diluent may be driven from the second chamber to the first chamber to form the lysate. The instrument 70 may control and/or drive the flow of the diluent from the second chamber to the first chamber. The lysate formed in the first chamber may contain the lysing agent, the diluent, and the whole blood sample. The diluent may be driven to the first chamber in advance of the arrival of the whole blood sample to prepare the lysate. Alternatively, the diluent and the whole blood sample may be driven to the first chamber simultaneously.

[0045] The filter 40 is in fluid communication with the lysing chamber and is configured to filter 40 the lysate into a filtered lysate. The filter 40 may filter out large, opaque structures from the lysate (e.g., hemoglobin) while allowing any genetic material from pathogen targets within the lysate to pass through the filter 40 for subsequent processing and analysis. In embodiments of the invention, the instrument 70 may control and/or drive the flow of the lysate from the lysing chamber and to through the filter 40 to form the filtered lysate. While FIG. 1 discloses a filter 40 downstream from the lysing chamber, a filter 40 may additionally or alternatively be provided upstream of the lysing chamber. Accordingly, in embodiments of the invention a filter 40 may be provided to filter the whole blood sample from the sampling device 20.

[0046] The meter 50 is in fluid communication with the filter 40 and is configured to meter a predetermined amount of filtered lysate for the NASBA analysis. The predetermined amount may, for example, be between 1 and 3 ml. In embodiments of the invention, the instrument 70 may control and/or drive the flow of the filtered lysate from the filter 40 and to the meter 50 to collect the predetermined amount of filtered lysate. While FIG. 1 discloses the meter 50 downstream from the lysing chamber and the filter 40, in embodiments of the invention an additional or alternative meter 50 may be provided upstream of the lysing chamber and/or of the filter 40. Accordingly, in embodiments of the invention a meter 50 may be provided to meter the whole blood sample and/or a filtered whole blood sample from the sampling device 20.

[0047] The NASBA fluidic network 60 may be in fluid communication with the meter 50 and may receive the predetermined amount of filtered lysate from the meter 50. The NASBA fluidic network 60 may include all of the materials (e.g., reagents, structures, etc.) necessary to perform predetermined NASBA-based nucleic-acid assays for mRNA and/or DNA on the predetermined amount of filtered lysate. The NASBA fluidic network 60 may include a plurality of reaction tubes that are each directly or indirectly in fluidic communication with the meter 50 and that are configured to receive filtered lysate from the meter 50. In embodiments of the invention, the instrument 70 may control and/or drive flow of the filtered lysate from the meter 50 to each of the plurality of reaction tubes.

[0048] Each of the plurality of reaction tubes may include all of the materials for processing the filtered lysate for isothermal amplification of predetermined genetic sequences of pathogen target (e.g., targeted mRNA to identify the presence of specific genes). Specific examples of materials that may be included in each of the plurality of reaction tubes include lysing buffers, mRNA-dependent DNA polymerase, mRNA primers, DNA primers, amino acids, and the like. Each of the plurality of reaction tubes may at least include an enzyme, a primer, and a beacon for performing an NASBA assay on a pathogen target within the filtered lysate. Each of the plurality of reaction tubes may include one or more of the following three enzymes: Avian Myeloblastosis Virus (AMV) Reverse Transcriptase, a Ribonuclease H (RNase H), and a T7 RNA polymerase. Each of the plurality of reaction tubes may include two or more oligonucleotide primers. The enzyme(s) and the primer(s) may amplify a predetermined genetic sequence in the pathogen target. The beacon provided in each of the plurality of reaction tubes may be configured to attach to the predetermined genetic sequence in the pathogen target. The beacon may include a fluorophore that emits light when attached to the predetermined genetic sequence of the pathogen target and when excited by an excitation source (e.g., a laser). Each reaction tube may include at least one window such that the instrument 70 may detect light emitted from the beacon when attached to a pathogen target. Each reaction tube may be provided with a beacon that is different from the beacons provided in each of the other reaction tubes. Accordingly, the NASBA fluidic network 60 may detect as many different predetermined pathogen targets as there are reaction tubes.

[0049] The NASBA fluidic network 60 may include a chamber containing an NASBA diluent. The chamber may be in fluid communication with each of the plurality of reaction tubes. In embodiments of the invention, the instrument 70 may control and/or drive flow of the diluent from the chamber to each of the plurality of reaction tubes. The diluent contained within the chamber may be fluidly communicated to each of the plurality of reaction tubes a predetermined period (e.g., 5 minutes) before introduction of the filtered lysate. After expiration of the predetermined period, the filtered lysate may be distributed to each of the plurality of reaction tubes to induce the NASBA reactions and the results of the NASBA reactions may be analyzed by the instrument 70.

[0050] The instrument 70 of the infection detection system 10 may be adapted to receive the sample processor 80, to initiate and/or control aspects of processing of the sample within the sample processor 80, and to analyze the processed sample. As discussed in detail above, the instrument 70 may control and/or drive fluid flow (e.g., whole blood flow, diluent flow, lysate flow, filtered lysate flow, etc.)· In addition, the instrument 70 may include a heater and/or a heat exchanger that may maintain the sample processor 80 within a predetermined temperature range necessary for isothermal amplification of predetermined pathogen targets during the NASBA assays. The predetermined temperature range may be within 35-50 degrees Celsius. In embodiments, the predetermined temperature range my be within 40 - 42 degrees Celsius.

[0051] In embodiments of the invention, the instrument 70 may be configured to perform any suitable NASBA-based nucleic-acid assay on the sample utilizing the reagents. For example, the instrument 70 may be configured to perform any steps for lysing pathogen cells and extracting and purifying pathogen messenger RNA (i.e., dissolve targeted mRNA and remove inhibitors that could interfere with nucleic acid amplification). In another example, the instrument 70 may be configured to perform any steps for processing the output solution from the extraction and purification steps for isothermal amplification of targeted mRNA to identify the presence of specific genes.

[0052] In an embodiment of the infection detection system 10, the infection detection system 10 includes a sampling device 20 configured to contain a whole blood sample containing a pathogen target. The infection detection system 10 includes a lysing chamber configured to be in fluid communication with the sampling device 20 to receive the whole blood sample, the lysing chamber being configured to lyse the whole blood sample into a lysate. The infection detection system 10 includes a filter 40 configured to be in fluid communication with the lysing chamber and to filter the lysate into a filtered lysate. The infection detection system 10 includes a meter configured to be in fluid communication with the filter 40 and configured to meter a predetermined amount of filtered lysate from the filtered lysate. The infection detection system 10 includes a NASBA fluidic network 60 configured to be in fluid communication with the meter to receive the predetermined amount of filtered lysate. The NASBA fluidic network 60 at least includes an enzyme and a primer for amplifying a predetermined genetic sequence in the pathogen target contained within the predetermined amount of filtered lysate; and a beacon that is configured to attach to the predetermined genetic sequence in the pathogen target. The infection detection system 10 include an instrument 70 configured to identify the beacon when the beacon is attached to the pathogen target to signal a presence of the pathogen target.

[0053] In an embodiment of the invention a method of detecting an infection using an infection detection system 10 in accordance with the above description includes collecting the whole blood sample containing the pathogen target, lysing the whole blood sample into the lysate, filtering the lysate into the filtered lysate, metering the predetermined amount of filtered lysate, amplifying the predetermined genetic sequence in the pathogen target contained within the predetermined amount of filtered lysate, attaching the beacon to the pathogen target; and exciting the beacon attached to the pathogen target to signal the presence of the pathogen target.

[0054] In an embodiment of the invention, an infection detection system 10 includes a sampling device 20 configured to contain a whole blood sample containing a pathogen target. The infection detection system 10 includes a sample processor 80 having a meter configured to be in fluid communication with the sampling device 20 and configured to meter a predetermined amount of the whole blood sample. The sample processor 80 includes a lysing chamber configured to be in fluid communication with the meter, the lysing chamber being configured to lyse the predetermined amount of the whole blood sample into a lysate. The sample processor 80 includes aNASBA fluidic network 60 configured to receive the lysate. The NASBA fluidic network 60 at least includes an enzyme and a primer for amplifying a predetermined genetic sequence in the pathogen target contained within the lysate, and a beacon that is configured to attach to the predetermined genetic sequence in the pathogen target. The infection detection system 10 includes an instrument 70 configured to identify the beacon when the beacon is attached to the pathogen target to signal a presence of the pathogen target.

[0055] The infection detection systems 10 may further include a filter 40 configured to be in fluid communication with the lysing chamber to filter the lysate prior to fluid communication of the lysate to the NASBA fluidic network 60.

[0056] The infection detection systems 10 in accordance with aspects of the invention may further includes further includes a whole blood sample contained within the sampling device 20 at a pressure lower than a pressure the sample processor 80 such that the whole blood sample is configured to be driven from the sampling device 20 to the conduits within the sample processor 80 when the sampling device 20 is connected to the sample processor 80.

[0057] The infection detection systems 10 in accordance with aspects of the invention may further include a sample processor 80 having an activator that is configured to activate fluid communication of the whole blood sample from the sampling device 20 to the sample processor 80. For example, as shown in FIGS. 2 - 7 the activator may be an air blister 206 that is configured to be in fluid communication with the sampling device 20 such that depression of the air blister 206 is configured to impart a positive pressure within the sampling device 20 to activate fluid communication of the whole blood sample from the sampling device 20 to the sample processor 80.

The sample processor 80 may include a sample tube port 200 having a first cannula 202 and a second cannula 204, the first cannula 202 being in fluid communication with the air blister 206 and the second cannula 204 being in fluid communication with the meter.

[0058] As shown in FIGS. 2-4 the air blister 206 may be located adjacent to the sample tube port 200. Further, a sample collection system (e.g. vacuum collection tube 400 or syringe) may attach directly to two types of access ports on a microfluidic cartridge. A syringe port 214 which is designed to accept a standard ISO Luer slip or Luer lock tipped syringe 402 and a sample collection tube port 200 which is designed to accept sample collection tubes 400 with form factor similar to vacuum collection tubes 400. These ports can be designed to have a snap lock mechanism or any such similar mechanism to hold and retain the sample collection system on the microfluidic cartridge. The port which accepts sample collection tubes 400 with form factor like vacuum collection tubes 400 also has two cannulas 202 and 204 which connect to the microfluidic network 60 on the microfluidic cartridge and pierce the seals on the sample collection tubes 400. An air blister 206 designed to displace a specified amount of air through outlet ports it connects to on the microfluidic chip is also incorporated on the microfluidic cartridge. The output ports connect to the microfluidic channel network on the cartridge. A metered sample well 208 on the microfluidic cartridge has fluidic connections to both the syringe port 214 and the vacuum tube sample collection port 200 with valves 212 in between the metered sample well 208 and the two access ports to control direction of flow of sample. The metered sample well 208 (i.e., a meter) is sealed off by a hydrophobic membrane 210 (e.g. include PTFE membranes) which acts as a natural stop for metering during transfer from the sample collection system connected to the vacuum collection tube port 200 or the syringe interface port 214. Activation of the blister by depressing it causes an exact amount of sample to be transferred from the vacuum collection tube 400 to the metering chamber. The metering chamber will have incorporated into it a visual indicator to signal completion of transfer of sample to it. The component(s) shown in FIGS. 2-4 and described above are intended to be used in an infection detection system 10, such as the infection detection system 10 shown schematically in FIG. 1 and described in detail above. Accordingly, the present invention includes an infection detection system 10 shown in FIG. 1 and described in detail above having any of the component(s) shown in FIGS. 2-4 and described above.

[0059] FIGS. 5-7 show an embodiment in which the air blister 206 is located within the sample tube port 200 and is configured to be depressed upon connection of the sampling device 20 and the sample tube port 200. That is, the air blister 206 is incorporated within the port that is designed to accept the sample collection tubes 400 with form factor like vacuum collection tubes 400. In this case the sample is transferred from the vacuum collection tube 400 to the metered sample well 208 in the process of attaching the vacuum tube sample collection system to the microfluidic cartridge. The component(s) shown in FIGS. 5-7 and described above are intended to be used in an infection detection system 10, such as the infection detection system 10 shown schematically in FIG. 1 and described in detail above. Accordingly, the present invention includes an infection detection system 10 shown in FIG. 1 and described in detail above having any of the component(s) shown in FIGS. 5-7 and described above.

[0060] FIGS. 8-10 show an embodiment of the invention wherein the instrument 70 and a cartridge interface 800 comprise the activator, and the instrument 70 is configured to impart a positive pressure, via the cartridge interface 800, within the sampling device 20 to activate fluid communication of the whole blood sample from the sampling device 20 to the sample processor 80. The sample processor 80 includes a sample tube port 200 having a first cannula 202 and a second cannula 204, the first cannula 202 being in fluid communication with the cartridge interface 800 and the second cannula 204 being in fluid communication with the meter. Further, the microfluidic cartridge has a port which interfaces to an instrument 70 and has a breakable seal incorporated in it. The instrument 70 breaks the seal on the cartridge and uses a positive pressure with a pressure sensor to control sample delivery from sample tube 400 to metered sample well 208. The pressure sensor incorporated in the instrument 70 enables detection of when the metered sample well 208 is full due to the change in pressure when sample contacts the hydrophobic membrane that seals the metered sample well 208. The component(s) shown in FIGS. 8-10 and described above are intended to be used in an infection detection system 10, such as the infection detection system 10 shown schematically in FIG. 1 and described in detail above. Accordingly, the present invention includes an infection detection system 10 shown in FIG. 1 and described in detail above having any of the component(s) shown in FIGS. 8-10 and described above.

[0061] FIGS. 11 and 12 show an embodiment of the invention wherein the activator is a piston integrated into the sample processor 80 and configured to selectively draw the whole blood sample from the sampling device 20. According to this embodiment, the sample processor 80 comprises a syringe port 214 in fluid communication with the meter for connecting with a syringe 402 and providing an alternative source of whole blood. The sample processor 80 further comprises a valve 212 for controlling fluid flow within the sample processor 80. The micro-fluidics cartridge will accept either a vacuum collection tube 400 or standard syringe 402 in order to receive the sample. In this embodiment, the instrument 70 transfers the sample from the vacuum collection tube 400 or syringe 402 into the cartridge. The person using the system need only connect the vacuum tube 400 or syringe 402 to the cartridge, and then insert the cartridge assembly into the instrument 70. The transfer/metering is accomplished by means of the piston integrated in the cartridge, which may be moved by the instrument 70 and may draw a precise amount of sample into the micro-fluidics cartridge, from either the vacuum tube 400 or syringe 402. Using the instrument 70 to transfer and meter the sample will allow for various metered sample volumes, if necessary, based on the test/cartridge being used. The instrument 70 may interrogate the cartridge in order to determine which cartridge/test is to be performed and set up parameters accordingly, including sample volume. The vacuum tube port may use a retainer designed in a way that will lock the vacuum tube 400 to the cartridge. The syringe port 214 is designed to accept a standard ISO Luer slip or Luer lock syringe 402. The vacuum tube 400/syringe 402 will stay attached to the cartridge through-out test and disposal, minimizing the potential risk of exposure to pathogens to the care giver. One-way valves may be employed in order to keep the metered sample from back flowing into the vacuum tube 400 or syringe 402. The component(s) shown in FIGS. 11 and 12 and described above are intended to be used in an infection detection system 10, such as the infection detection system 10 shown schematically in FIG. 1 and described in detail above. Accordingly, the present invention includes an infection detection system 10 shown in FIG. 1 and described in detail above having any of the component(s) shown in FIGS. 11 and 12 and described above.

[0062] As shown in FIG. 13, the infection detection systems 10 of the present invention may further include a sampling device 20 that is a blood press syringe 402. The blood press syringe 402 may include a plunger having a seal, a barrel including a cavity configured to receive the whole blood sample and to slidingly receive the plunger, a filter 1300 comprising wire mesh attached to the barrel and configured to separate clear liquids from proteins and solids in a whole blood sample when the plunger forces the whole blood sample through the filter 1300. The component(s) shown in FIG. 13 and described above is intended to be used in an infection detection system 10, such as the infection detection system 10 shown schematically in FIG. 1 and described in detail above. Accordingly, the present invention includes an infection detection system 10 shown in FIG. 1 and described in detail above having any of the component(s) shown in FIG. 13 and described above.

[0063] Embodiments of the invention may further include a method of detecting an infection using any of the above described infection detection systems 10. The method may include collecting the whole blood sample containing the pathogen target, metering the predetermined amount of the whole blood sample, lysing the metered whole blood sample into the lysate, filtering the lysate into the filtered lysate, amplifying the predetermined genetic sequence in the pathogen target contained within the predetermined amount of filtered lysate, attaching the beacon to the pathogen target, and exciting the beacon attached to the pathogen target to signal the presence of the pathogen target.

[0064] Aspects of the various embodiments of the invention are further described in FIGS. 1-13.

[0065] The many features and advantages of the infection detection system 10 and methods described herein are apparent from the detailed specification, and thus, the claims cover all such features and advantages within the scope of this application. Further, numerous modifications and variations are possible. As such, it is not desired to limit the infection detection system 10 to the exact construction and operation described and illustrated and, accordingly, all suitable modifications and equivalents may fall within the scope of the claims.

Claims

Claims What is claimed is:

1. An infection detection system (10) comprising: a sampling device (20) configured to contain a whole blood sample containing a pathogen target; a lysing chamber configured to be in fluid communication with the sampling device (20) to receive the whole blood sample, the lysing chamber being configured to lyse the whole blood sample into a lysate; a filter (40) configured to be in fluid communication with the lysing chamber and to filter the lysate into a filter lysate; a meter (50) configured to be in fluid communication with the filter (40) and configured to meter a predetermined amount of filtered lysate from the filtered lysate; a NASB A fluidic network (60) configured to be in fluid communication with the meter (50) to receive the predetermined amount of filtered lysate, the NASB A fluidic network (60) comprising: an enzyme and a primer for amplifying a predetermined genetic sequence in the pathogen target contained within the predetermined amount of filtered lysate; and a beacon that is configured to attach to the predetermined genetic sequence in the pathogen target; and an instrument (70) configured to identify the beacon when the beacon is attached to the pathogen target to signal a presence of the pathogen target.

2. A method of detecting an infection using the infection detection system (10) as claimed in claim 1, the method comprising: collecting the whole blood sample containing the pathogen target; lysing the whole blood sample into the lysate; filtering the lysate into the filtered lysate; metering the predetermined amount of filtered lysate; amplifying the predetermined genetic sequence in the pathogen target contained within the predetermined amount of filtered lysate; attaching the beacon to the pathogen target; and exciting the beacon attached to the pathogen target to signal the presence of the pathogen target.

3. An infection detection system (10) comprising: a sampling device (20) configured to contain a whole blood sample containing a pathogen target; a sample processor (80) comprising: a meter (50) configured to be in fluid communication with the sampling device (20) and configured to meter a predetermined amount of the whole blood sample; a lysing chamber configured to be in fluid communication with the meter (50), the lysing chamber being configured to lyse the predetermined amount of the whole blood sample into a lysate; aNASBA fluidic network (60) configured to receive the lysate, the NASBA fluidic network (60) comprising: an enzyme and a primer for amplifying a predetermined genetic sequence in the pathogen target contained within the lysate; and a beacon that is configured to attach to the predetermined genetic sequence in the pathogen target; and an instrument (70) configured to identify the beacon when the beacon is attached to the pathogen target to signal a presence of the pathogen target.

4. The infection detection system (10) of claim 3, further comprising a filter (40) configured to be in fluid communication with the lysing chamber to filter the lysate prior to fluid communication of the lysate to the NASBA fluidic network (60).

5. The infection detection system (10) according to any of the preceding claims, wherein the whole blood sample is contained within the sampling device (20) at a pressure lower than a pressure the sample processor (80) such that the whole blood sample is configured to be driven from the sampling device (20) to the conduits within the sample processor (80) when the sampling device (20) is connected to the sample processor (80).

6. The infection detection system (10) according to any of the preceding claims, wherein the sample processor (80) includes an activator that is configured to activate fluid communication of the whole blood sample from the sampling device (20) to the sample processor (80).

7. The infection detection system (10) according to claim 6, wherein the activator is an air blister (206) that is configured to be in fluid communication with the sampling device (20) such that depression of the air blister (206) is configured to impart a positive pressure within the sampling device (20) to activate fluid communication of the whole blood sample from the sampling device (20) to the sample processor (80).

8. The infection detection system (10) according to claim 7, wherein the sample processor (80) includes a sample tube port (200) having a first cannula (202) and a second cannula (204), the first cannula (202) being in fluid communication with the air blister (206) and the second cannula (204) being in fluid communication with the meter (50).

9. The infection detection system (10) according to claim 8, wherein the air blister (206) is located adjacent to the sample tube port (200).

10. The infection detection system (10) according to claim 7, wherein the air blister (206) is located within the sample tube port (200) and is configured to be depressed upon connection of the sampling device (20) and the sample tube port (200).

11. The infection detection system (10) according to claim 6, wherein the instrument (70) and a cartridge interface (800) comprise the activator, and the instrument (70) is configured to impart a positive pressure, via the cartridge interface (800), within the sampling device (20) to activate fluid communication of the whole blood sample from the sampling device (20) to the sample processor (80).

12. The infection detection system (10) according to claim 11, wherein the sample processor (80) includes a sample tube port (200) having a first cannula (202) and a second cannula (204), the first cannula (202) being in fluid communication with the cartridge interface (800) and the second cannula (204) being in fluid communication with the meter (50).

13. The infection detection system (10) according to claim 6, wherein the activator is a piston integrated into the sample processor (80) and configured to selectively draw the whole blood sample from the sampling device (20).

14. The infection detection system (10) according to any of the preceding claims, wherein the sample processor (80) comprises a syringe port (214) in fluid communication with the meter (50) for connecting with a syringe (402) and providing an alternative source of whole blood.

15. The infection detection system (10) according to any of the preceding claims, wherein the sample processor (80) comprises a valve (212) for controlling fluid flow within the sample processor (80).

16. The infection detection system (10) according to any of the preceding claims, further comprising a blood press syringe (402) comprising: a plunger having a seal; a barrel including a cavity configured to receive the whole blood sample and to slidingly receive the plunger; a filter (40, 1300) comprising wire mesh attached to the barrel and configured to separate clear liquids from proteins and solids in a whole blood sample when the plunger forces the whole blood sample through the filter (40, 1300).

17. The infection detection system (10) according to any of claims 1 and 3-16, wherein the primer includes any one or more of oligonucleotide sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 93, and SEQ ID NO: 94.

18. The infection detection system (10) according to any of claims 1 and 3-17, wherein the beacon includes any one or more of oligonucleotide sequences SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 31, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 47, SEQ ID NO: 51, SEQ ID NO: 55, SEQ ID NO: 59, SEQ ID NO: 63, SEQ ID NO: 67, SEQ ID NO: 71, SEQ ID NO: 75, SEQ ID NO: 79, SEQ ID NO: 83, SEQ ID NO: 87, SEQ ID NO: 91, and SEQ ID NO: 95.

19. The infection detection system (10) according to any of claims 1 and 3-18, wherein the lysing chamber comprises a lysis solution for lysing microorganisms in the whole blood sample, the lysis solution comprising:

2 mM to 16 mM of a Quanidinium thiocyanate/guanidine thiocyanate;

20 mM to 160 pM of a Tris HCL, pH 8.5;

6 pM to 48 pM of a Magnesium chloride;

35 pM to 280 pM of a Potassium chloride; and

0.1% v/v to 1.0% v/v of an octylphenoxypolyethoxyethanol.

20. A method of detecting an infection using the infection detection system (10) as claimed in any of claims 3-18, the method comprising: collecting the whole blood sample containing the pathogen target; metering the predetermined amount of the whole blood sample; lysing the metered whole blood sample into the lysate; filtering the lysate into the filtered lysate; amplifying the predetermined genetic sequence in the pathogen target contained within the predetermined amount of filtered lysate; attaching the beacon to the pathogen target; and exciting the beacon attached to the pathogen target to signal the presence of the pathogen target.

21. The method of detecting an infection using the infection detection system (10) as claimed in claim 2 or claim 20, wherein the lysing step is accomplished using the lysis solution comprising:

2 mM to 16 mM of a Quanidinium thiocyanate/guanidine thiocyanate;

20 pM to 160 pM of a Tris HCL, pH 8.5;

6 pM to 48 pM of a Magnesium chloride;

35 pM to 280 pM of a Potassium chloride; and

0.1% v/v to 1.0% v/v of an octylphenoxypolyethoxyethanol.