Systems, methods and devices for concentrating and identifying microorganisms from blood

Abstract

The present invention relates to systems, methods and devices for isolating and identifying microorganisms from samples known to or capable of containing microorganisms.

Description

[Technical field]

[0001] Related Applications This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 126,041, filed December 16, 2020, the entirety of which is incorporated herein by reference.

[0002] 1.Technical Field SUMMARY OF THE DISCLOSURE Embodiments of the present disclosure generally relate to systems, methods, and devices for sepsis diagnosis directly from blood. [Background technology]

[0003] 2.Background In the United States, Canada, and Western Europe, infectious diseases account for approximately 7% of human mortality, while in developing regions, infectious diseases account for more than 40% of human mortality. Infectious diseases cause a variety of clinical symptoms. Common overt signs include fever, pneumonia, meningitis, diarrhea, and diarrhea containing blood. While physical signs suggest some pathogens and exclude others as pathogens, various causative agents remain possible, and a definite diagnosis often requires the performance of various assays.

[0004] In the United States, bloodstream infections (BSIs) and the resulting septic shock (also referred to as sepsis, septicemia, bacteremia, fungemia, candidiasis, candidemia, blood-borne infections, and other related terms) are a leading cause of death. For example, bacterial BSIs are the 11th leading cause of death in adults and the 7th leading cause of death in young children. Candida spp. and other fungi can also cause BSIs. Candida spp. bloodstream infections are associated with a high mortality rate (40%), primarily attributable to the long diagnostic time required for blood cultures. Studies have shown that initiation of appropriate antibacterial or antifungal therapy can reduce mortality, and that each time antibiotic administration is delayed, there is a significant increase in mortality. Therefore, early detection and definitive diagnosis, as well as prompt treatment with appropriate antibiotics or antifungals, are desirable to improve outcomes for patients with suspected BSIs.

[0005] The current diagnostic gold standard for BSI requires growth of the organism in culture followed by microscopic observation, subculture, and phenotypic identification of purified isolates. This results in reporting times ranging from 36-72 hours for gram-positive bacteria, 48-96 hours for gram-negative bacteria, and 48-120 hours for fungal infections. Surprisingly, approximately one-third of patients treated for fungal BSI never show positive blood culture growth, and many positive cases of fungal BSI are definitively diagnosed solely based on postmortem analysis. As a result, it is typical for physicians to begin treatment of patients suspected of having a BSI with a broad-spectrum antibiotic or antifungal regimen immediately after blood is drawn for culture. This is not ideal. Studies have shown that administration of inappropriate or ineffective antimicrobial therapy does not help improve patient outcomes and, troublingly, has been found to cause an increase in drug-resistant organisms, independently compromising patient outcomes and public health overall.

[0006] One alternative to the diagnostic gold standard (i.e., classical microbiological methods) involves molecular identification of infectious bacteria and fungi from blood. However, the number of infectious organisms found in whole blood in BSI is usually low (approximately 1-100 colony forming units (cfu / ml) per mL of blood, with approximately 1-10 cfu / ml being typical in most individuals with culture-confirmed sepsis). Furthermore, blood contains several inhibitors of polymerase chain reaction (PCR) (e.g., hemoglobin and other blood proteins (e.g., human serum albumin) and genomic DNA: from leukocytes that may co-purify with the organism and interfere with both nucleic acid recovery from the target organism and downstream PCR). Due to such low abundance of organisms in whole blood and the presence of PCR inhibitors, enrichment from larger volumes of whole blood (e.g., 1-20 mL) is required to obtain the quality and quantity of DNA template desired to achieve sensitivity at clinically relevant organism levels.

[0007] There is an urgent need for faster and more accurate molecular-based diagnostics to reduce the number of ineffective or unnecessary broad-spectrum antibiotics that non-infected patients receive. Rapid diagnostics could allow for more tailored and timely administration of effective antimicrobial therapy to patients who do have a BSI. Despite these many potential benefits, many of the attempted rapid BSI diagnostic solutions have not been widely adopted. This is due to a variety of reasons, including cumbersome workflows, time to results, and cost.

[0008] One product available on the market is called MolYsis™, which offers the promise of selective isolation of bacterial DNA from intact organisms in whole blood. The MolYsis™ Complete5 DNA Extraction Kit (catalog number D-321-100; Molzym GmbH&Co.KG, Bremen, Germany) contains a chaotropic lysis buffer for selective lysis of blood factors (red blood cells, white blood cells, etc.) and DNase to degrade genomic DNA. The microbial cells are collected by centrifugation, the supernatant is discarded, the cells are resuspended several times in different buffers, re-pelleted, chemical lysis of the microbial cells is performed, and finally the microbial nucleic acid is recovered. All in all, the MolYsis™ kit involves a cumbersome workflow that takes about 45 minutes for sample preparation, plus another 45 minutes for purification and lysis of the microbial cells. Identification of the microorganism requires inputting the nucleic acid recovered with the MolYsis™ kit into another assay that takes additional time and involves additional expenditure. Furthermore, successful use of the MolYsis™ kit requires skilled personnel. Dependence on operator skill introduces the risk that the yield and quality of results will vary from operator to operator. The many buffers and manual pipetting steps increase the risk of cross-contamination of samples.

[0009] Another product intended for use in identifying BSIs from whole blood is the T2MR® from T2 Biosystems. The T2MR® system includes automated sample preparation including selective lysis of blood factors, microbial recovery, microbial lysis, recovery of microbial-derived nucleic acids, and PCR amplification. The T2MR® system uses nuclear magnetic resonance (NMR) for microbial identification. Superparamagnetic NMR nanoprobes in solution bind to DNA specific to microorganisms and form aggregates that are detectable by NMR. Nanoprobes that are aggregated by the presence of microbial nucleic acids generate a larger NMR signal compared to the signal from non-aggregated nanoprobes. However, the T2MR® system requires 4-6 hours of NMR data collection to obtain data of sufficient quality that can be used for microbial identification. Additionally, the T2MR® system is expensive (the device costs approximately $150,000) and the throughput of the device is significantly limited due to the time required for data collection. Additionally, bacteria and fungi associated with BSIs are tested against separate T2MR® panels. This means that any patient presenting with sepsis will require testing against bacterial and fungal panels to include / rule out bacterial and fungal causes. Furthermore, the number of organisms tested against bacterial and fungal panels is limited (approximately 6 organisms tested for each) and the tests do not provide information about drug susceptibility / resistance.

[0010] The present invention addresses a variety of improvements regarding identification of BSI-associated microorganisms directly from blood, with a simplified workflow and faster sample-to-answer. Summary of the Invention [Means for solving the problem]

[0011] The present invention provides methods, systems, and devices for concentrating, characterizing, and / or identifying microorganisms from a sample. In one embodiment, the microorganism is a bacterium. In another embodiment, the microorganism is a fungal organism (e.g., a yeast or mold). In a further embodiment, the microorganism is a parasite. The methods, systems, and devices may be particularly useful for isolating, concentrating, characterizing, and / or identifying microorganisms from complex samples such as blood or urine or cerebrospinal fluid. In a preferred aspect, the methods, systems, and devices of the present invention may be used for concentrating, characterizing, and / or identifying microorganisms directly from whole blood to rapidly determine whether a patient is septic. In typical sepsis, the concentration of microorganisms in the bloodstream is low, e.g., < about 1-100 cfu / ml, with < about 1-10 cfu / ml being typical. In one or more septic patients suspected of having sepsis, the microorganisms in the blood, if any, are very dilute and therefore identified directly from the blood sample without using the methods described herein. Furthermore, blood contains several inhibitors of PCR (e.g., hemoglobin, human serum albumin, and genomic DNA) that may be preferably removed to ensure consistent successful identification and analysis of microorganisms from whole blood and other complex matrices (e.g., urine and CSF).The present invention provides methods, systems, and devices for selective lysis of non-microbial cells in a sample and concentration of microorganisms from a relatively large volume (e.g., 10-20 ml) of the sample.In preferred embodiments, the methods, systems, and devices described herein do not include the use of devices or steps such as, but not limited to, mixing a blood sample and a differential lysis buffer in a first container and then transferring the lysate to a centrifugal concentrator containing components other than the blood sample and the differential lysis buffer in the centrifugal concentrator, opening the centrifugal concentrator after centrifugation and decanting the supernatant fraction, recovering microorganisms by centrifugation with a high density cushion or physical separator, pre-treating the blood sample (other than mixing the blood sample with a differential lysis buffer and processing it with the concentration and identification steps described in the methods herein), preliminary analysis and incubation steps, subculturing the sample and identifying microorganisms present in the sample, or a DNase step to selectively digest non-microbial DNA from the lysed non-microbial cells.

[0012] The invention described herein may preferably include a method for isolating and identifying the described microorganisms. The method may preferably include the steps of: (a) providing a volume of a blood sample suspected of containing the microorganism; (b) mixing the blood sample with a differential lysis buffer to obtain a lysate containing lysed hemocytes and non-lysed microorganisms; (c) concentrating the microorganisms from the lysate; (d) adding the microorganisms to a device containing one or more reagents required for identifying the microorganism; and (e) identifying the microorganisms present in the blood sample. In the method, the microorganisms, if present, are concentrated in the range of 25-100 fold compared to the volume of the provided blood sample, and the microorganisms, if present, have a concentration in the provided blood sample in the range of < about 1 CFU / ml (but greater than 0) to about 100 CFU / ml (e.g., < 1 CFU / ml 10 to about 10 CFU / ml).

[0013] Method steps (a)-(c) may preferably be completed within a time range of about 10-20 minutes, and method steps (d) and (e) may preferably be completed within a time range of less than 4 hours, less than 3 hours, less than 2 hours, or less than 1 hour.

[0014] The microorganism in the method may suitably include one or more bacterial or fungal organisms associated with blood borne infections.

[0015] The identification in the method may preferably include one or more of molecular, phenotypic, proteomic, optical, or culture-based testing. The identification may preferably include isolating one or more nucleic acids characteristic of the microorganism from the microorganism, and analyzing the one or more nucleic acids to identify the microorganism present in the blood sample. In one embodiment of the method, the identification further includes amplifying the one or more nucleic acids and then detecting the one or more amplified nucleic acids. The detection of the one or more amplified nucleic acids may preferably include one or more of the use of a dsDNA binding dye, real-time PCR, a post-amplification nucleic acid melting step, a nucleic acid sequencing step, a labeled DNA binding probe, or an unlabeled probe. The identifying step may preferably be completed within a time range of about 5 to 75 minutes.

[0016] The method may suitably further comprise carrying out a culturing step on the concentrated microorganism in the medium to increase the concentration of the microorganism and then carrying out an identifying step, wherein the culturing step is carried out for 4 hours or less, 3 hours or less, or 2 hours or less, 1 hour or less, 30 minutes or less, 20 minutes or less, or 10 minutes or less, preferably 3 hours or less.

[0017] The differential lysis buffer used in the recited methods may preferably include a buffer substance, a non-ionic surfactant, a salt, and a pH range of about 10-11 before mixing the blood sample with the differential lysis buffer. The differential lysis buffer may preferably have a pH of about 7.0-8.0 after mixing the blood sample with the differential lysis buffer. The buffer substance used in the differential lysis buffer may preferably be selected from the group consisting of CABS, CAPS, CAPS, CHES, and combinations thereof. The buffer substance used in the differential lysis buffer may preferably be CAPS. The pH of the differential lysis buffer mixed with the blood sample may preferably be about 1.5-2.5 pH units below the pH buffer range of the buffer substance. The non-ionic surfactant used in the differential lysis buffer may suitably be one or more of polyoxyethylene (POE) ethers, preferably Arlasolve 200 (also known as poly(oxy-1,2-ethanediyl)), BrijO10, and nonaethylene glycol monododecyl ether (also known as Brij 35). The non-ionic surfactant used in the differential lysis buffer may suitably be selected from the group consisting of Triton X-114, NP-40, Arlasolve 200, BrijO10 (also known as Brij 96 / 97), octyl β-D-glucopyranoside, saponin, nonaethylene glycol monododecyl ether (also known as Brij 35), and combinations thereof. In the differential lysis buffer that is combined with the blood sample, the detergent concentration (e.g., in the range of 0.1%-0.5%) and pH (e.g., in the range of 7-11) may be adjusted to maximize differential lysis of blood cells in the sample while minimizing pellet volume. Preferably, the pellet volume may be about 500 μL or less, about 400 μL or less, about 300 μL or less, about 200 μL or less, or about 100 μL or less. Preferably, up to 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% of the non-microbial cells in the sample may be lysed within 2-5 minutes of combining the sample with the differential lysis buffer.

[0018] Concentrating the microorganisms from the lysate may suitably include centrifugation, and the concentration further comprises recovering a pellet fraction comprising the microorganisms from a supernatant fraction comprising the lysed blood fraction. Concentrating the microorganisms from the lysate may suitably include placing the blood sample mixed with the differential lysis buffer in a centrifugal concentrator; centrifuging the centrifugal concentrator to concentrate the microorganisms from the blood sample placed in the chamber; and causing the concentrated microorganisms to emerge from a second opening at a second end of the chamber, where the centrifugal concentrator includes a chamber having an opening at a first end and a sealing portion at a second end, the sealing portion designed to seal the second opening at the second end of the chamber; and a plunger at least partially movably disposed inside the chamber, the plunger designed to actuate to open the sealing portion. Expressing the concentrated microorganisms from the second opening at the second end of the chamber may suitably include aseptically causing the pellet to emerge from the second end of the centrifugal concentrator into a vial or assay device. The centrifugal concentrator may preferably not include a high density cushion or physical separator to separate the microorganisms from the lysate.

[0019] The method may preferably not include one or more of: mixing the blood sample and differential lysis buffer in a first container and then transferring the lysate to a centrifugal concentrator containing components other than the blood sample and differential lysis buffer in the centrifugal concentrator, opening the centrifugal concentrator after centrifugation and decanting the supernatant fraction, an incubation step before mixing the blood sample with the differential lysis buffer, or a DNase step to digest genomic DNA in the lysate.The method may preferably not include one or more of an incubation step before mixing the blood sample with the differential lysis buffer, or a DNase step to digest genomic DNA in the lysate.

[0020] The microorganisms may be suitably concentrated from the lysate by filtration techniques, and the method may suitably further comprise adding the filter having the concentrated microorganisms thereon to one or more culture or assay devices designed to identify microorganisms present in the blood sample.

[0021] Preferably, the method of mixing the blood sample with the differential lysis buffer to obtain a lysate and separating the microorganisms from the lysate may be accomplished in a single tube. Preferably, the differential lysis buffer used in the method may be a single buffer provided in a single tube. Preferably, the differential lysis buffer used in the method may be free of DNase or protease, and the method may preferably not include the step of adding exogenous DNase or protease to the single tube. Preferably, the differential lysis buffer used in the method may be compatible with an anticoagulant selected from the group consisting of EDTA, citrate, acid citrate dextrose (ACD) sodium polyanethole sulfate (SPS), heparan, sodium fluoride / oxalate, and combinations thereof.

[0022] The invention described herein may suitably include a method for concentrating and identifying microorganisms from blood, which may suitably include the steps of: (a) providing a blood sample known or capable of containing microorganisms; (b) mixing the blood sample with a differential lysis buffer comprising a buffer substance, a non-ionic surfactant, and a salt to obtain a lysate comprising lysed hemocytes and unlysed microorganisms by mixing; (c) concentrating the microorganisms from the lysate; and (d) identifying the microorganisms present in the blood sample, wherein the blood sample mixed with the differential lysis buffer has a pH of about 7.0-8.0, and the buffer substance has a useful pH buffer range of about 8.6-11.4, wherein the microorganisms are concentrated within a range of 25-100 fold relative to the starting volume of the provided blood sample, and wherein identification is accomplished in 4 hours or less, 3 hours or less, 2 hours or less, or 1 hour or less.

[0023] Identification may suitably include one or more of molecular, phenotypic, proteomic, optical, or culture-based testing. The identifying step of the method may suitably include isolating one or more nucleic acids characteristic of the microorganism from the microorganism, and analysing the one or more nucleic acids to identify the microorganism present in the blood sample.

[0024] The non-ionic surfactant recited in the method may suitably be one or more of polyoxyethylene (POE) ethers, preferably Arlasolve 200 (also known as poly(oxy-1,2-ethanediyl)), BrijO10, and nonaethylene glycol monododecyl ether (also known as Brij 35). The non-ionic surfactant recited in the method may suitably be selected from the group consisting of Triton X-114, NP-40, Arlasolve 200, BrijO10 (also known as Brij 96 / 97), octyl β-D-glucopyranoside, saponin, nonaethylene glycol monododecyl ether (also known as Brij 35), and combinations thereof.

[0025] The buffer recited in the method may be preferably selected from the group consisting of CABS, CAPS, CAPSO, CHES, and combinations thereof. The buffer recited in the method may be preferably CAPS, which has a pH buffering range of about 9.7 to 11.1 and a pKa of about 10.4 at 25°C.

[0026] The detergent concentration (e.g., in the range of 0.1%-0.5%) and pH (e.g., in the range of 7-11) may be adjusted to maximize differential lysis of blood cells in the sample while minimizing pellet volume. Preferably, the pellet volume may be about 500 μL or less, about 400 μL or less, about 300 μL or less, about 200 μL or less, or about 100 μL or less. Preferably, up to 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% of the non-microbial cells in the sample may be lysed within 2-5 minutes of combining the sample with the differential lysis buffer.

[0027] The salt recited in the method may suitably be sodium chloride.

[0028] The method may preferably not include a blood culture step prior to enrichment and / or a DNase step to digest genomic DNA in the lysate.

[0029] Steps (a)-(c) of the method may preferably be completed within a time range of about 10-20 minutes. The isolating and analyzing steps of the method may preferably be completed within a time range of about 5-75 minutes. The time to obtain the lysate may preferably be in the range of about 2-10 minutes, preferably about 3-5 minutes. The generation of the lysate may preferably not include additional steps other than combining (i.e., combining the blood sample with the differential lysis buffer and incubating the blood / buffer mixture for a time sufficient to lyse the blood cells in the sample (about 2-10 minutes, preferably about 3-5 minutes).

[0030] The following is what will be explained:

[0031] A1. A method for isolating and identifying microorganisms, comprising: (a) providing a blood sample known to contain or capable of containing a microorganism; (b) mixing the blood sample with a differential lysis buffer having a pH to obtain a lysate containing lysed hemocytes and unlysed microorganisms; (c) isolating the microorganisms from the lysate; and (d) adding the microorganism to an assay device that contains one or more reagents required to identify the microorganism; (e) identifying microorganisms present in the blood sample; and wherein identifying comprises isolating from the microorganism one or more nucleic acids characteristic of the microorganism and analyzing the one or more nucleic acids to identify the microorganism present in the blood sample.

[0032] A2. The method of clause A1, wherein the microorganism is one or more bacteria or yeasts associated with blood-borne infections.

[0033] A3. The method of clause A1 and / or clause A2, further comprising first identifying one or more symptoms of sepsis, septic infection, septic shock, septicemia, etc. in the patient to determine that the patient has a blood-borne infection.

[0034] A4. One or more of the methods of clauses A1-A3, wherein the differential lysis buffer comprises a buffering agent, a non-ionic surfactant, and a pH range of about 10-11 before mixing the blood sample with the differential lysis buffer and a pH of about 7.0-8.0 after mixing the blood sample with the differential lysis buffer.

[0035] A5. The method of any one of clauses A1-A4, wherein the non-ionic surfactant is a polyoxyethylene (POE) ether.

[0036] A6. One or more of the methods of clauses A1-A5, wherein the non-ionic surfactant is selected from the group consisting of Triton X-114, NP-40, Arlasolve 200, BrijO10 (also known as Brij 96 / 97), octyl β-D-glucopyranoside, saponin, nonaethylene glycol monododecyl ether (C12E9, polidocenol), and combinations thereof.

[0037] A7. One or more of the methods of clauses A1-A6, wherein the separation of the microorganisms from the lysate comprises a centrifugation step, and the separation further comprises recovering a pellet fraction comprising the microorganisms from a supernatant fraction comprising the lysed blood fraction.

[0038] A8. placing the blood sample mixed with the differential lysis buffer into a centrifugal concentrator; centrifuging the centrifugal concentrator to pellet microorganisms from the blood sample disposed within the chamber; depressing the plunger to open the seal and cause the pellet to emerge from an opening at the second end of the chamber; wherein the centrifugal concentrator further comprises: a chamber having an open portion at a first end and a sealing portion at a second end, the sealing portion configured to seal the second open portion at the second end of the chamber; and a plunger movably disposed at least partially inside the chamber, the plunger designed to be actuated to open the seal; one or more of the methods of clauses A1 to A7,

[0039] A9. The method of one or more of clauses A1-A8, wherein depressing the plunger, opening the seal portion, and producing a pellet includes depressing the plunger into sealing engagement with a portion of the body, and expelling the pellet from the second end by opening the seal under pressure.

[0040] A10. The method of any one or more of clauses A1-A9, further comprising forming a pellet from the second end of the centrifugal concentrator into a vial or a self-contained assay device.

[0041] A11. The method of any one of clauses A1-A10, wherein each of the centrifugal concentrator and the vial is designed to couple the second end of the centrifugal concentrator to the vial.

[0042] A12. The method of any one of clauses A1-A11, wherein the vial is designed to deliver the pellet to a self-contained molecular analysis device.

[0043] A13. The method of any one or more of clauses A1-A12, wherein the vial is designed to deliver the pellet to a self-contained molecular analysis device without decoupling the vial from the second end of the centrifugal concentrator.

[0044] A14. Any one or more of the methods of clauses A1-A13, wherein the opening at the first end of the centrifugal concentrator includes a septum, similarly functional structure, or the like, designed to aseptically load the sample mixed with the differential lysis buffer into the centrifugal concentrator.

[0045] A15. The method of one or more of clauses A1-A14, wherein the centrifugal concentrator does not include a high density cushion or a physical separator.

[0046] A16. The method of any one or more of clauses A1-A15, which does not include one or more of: mixing the blood sample and the differential lysis buffer in a first container and then transferring the lysate to a centrifugal concentrator containing components other than the blood sample and the differential lysis buffer in the centrifugal concentrator; opening the centrifugal concentrator after centrifugation and decanting the supernatant fraction; pre-treating the blood sample; a blood culturing step; a step of subculturing the blood sample to identify microorganisms present in the blood sample; or a DNase step to digest genomic DNA in the lysate.

[0047] A17. One or more of the methods of clauses A1-A16, not including one or more of the following: pre-treating the blood sample, a blood culture step, a step of subculturing the blood sample and identifying microorganisms present in the blood sample, or a DNase step for digesting genomic DNA in the lysate.

[0048] A18. The method of any one or more of clauses A1-A17, wherein steps (a)-(c) are completed within a time range of about 10-20 minutes.

[0049] A19. The method of any one or more of clauses A1-A18, wherein steps (d) and (e) are completed within a time range of about 15-75 minutes.

[0050] A20. Steps (a)-(e) are completed within a time range of about 25-95 minutes.

[0051] A21. The method of any one of clauses A1 to A20, wherein the microorganisms are separated from the lysate by a filter.

[0052] A22. The method of any one of clauses A1-A21, further comprising adding a filter to a self-contained assay device.

[0053] A23. The method of any one of clauses A1-A22, wherein the differential lysis buffer comprises a buffer substance having a pH buffer range, and the pH of the differential lysis buffer mixed with the blood sample is outside the pH buffer range of the buffer substance.

[0054] A23.1 The method of one or more of clauses A1-A23, wherein the buffering substance is selected from the group consisting of CABS, CAPS, CAPS, CHES, and combinations thereof.

[0055] A23.2 The method of one or more of clauses A1 to A23.1, wherein the buffer substance is CAPS.

[0056] A24. One or more of the methods of clauses A1 to A23.2, in which the pH of the differential lysis buffer mixed with the blood sample is below the pH buffer range of the buffer substance.

[0057] A25. One or more of the methods of clauses A1-A24, wherein the pH of the differential lysis buffer mixed with the blood sample is about 1.5-2.5 pH units below the pH buffering range of the buffering substance.

[0058] A26. The method of any one of clauses A1-A25, wherein the blood sample mixed with the differential lysis buffer has a pH of about 7.0-8.0, and the buffering substance has a useful pH buffering range of about 8.6-11.4 and a pKa within the range of about 9.5 to about 10.7 at 25°C.

[0059] A27. One or more of the methods of clauses A1-A26, wherein the identifying further comprises amplifying one or more nucleic acids and then detecting one or more amplified nucleic acids.

[0060] A28. One or more methods of clauses A1 to A27, wherein the detection of one or more amplified nucleic acids comprises a nucleic acid melting step.

[0061] A29. One or more of the methods of clauses A1-A28, further comprising: performing a first-stage multiplex amplification of one or more nucleic acids to obtain first-stage amplification products; separating the diluted first-stage amplification products in a set of second-stage amplification wells, each of which has an amplification primer set designed to further amplify a specific nucleic acid that may be present in the sample; performing a second-stage amplification in the second-stage amplification wells; and performing a post-amplification nucleic acid melt and melting curve analysis to identify microorganisms present in the blood sample.

[0062] A30. One or more of the methods of clauses A1-A29, wherein the analysis includes a nucleic acid sequencing step to generate sequencing data comprising sequence information obtained from one or more nucleic acids sufficient to identify microorganisms present in the blood sample.

[0063] A31. One or more methods of clauses A1-A30, wherein the nucleic acid sequencing step comprises massively parallel or next-generation sequencing techniques.

[0064] A32. One or more of the methods of clauses A1-A31, wherein the steps of mixing the blood sample with a differential lysis buffer to obtain a lysate and isolating the microorganisms from the lysate are accomplished in a single tube.

[0065] A33. The method of any one of clauses A1-A32, wherein the differential lysis buffer is a single buffer provided in a single tube.

[0066] A34. The method of one or more of clauses A1-A33, wherein the differential lysis buffer does not contain DNase or protease and the method does not include the step of adding exogenous DNase or protease to a single tube.

[0067] A35. One or more of the methods of clauses A1-A34, wherein the differential lysis buffer is compatible with standard anticoagulants, such as, but not limited to, those selected from the group consisting of EDTA, citrate, sodium polyanethol sulfate (SPS), heparan, sodium fluoride / oxalate, and combinations thereof.

[0068] B1. A method for isolating and identifying microorganisms, comprising: (a) providing a blood sample known to contain or capable of containing a microorganism; (b) mixing the blood sample with a differential lysis buffer comprising a buffer substance and a non-ionic surfactant, thereby obtaining a lysate comprising lysed hemocytes and unlysed microorganisms; (c) isolating the microorganisms from the lysate; and (d) adding the microorganism to a self-contained assay device designed to perform an assay involving amplification of one or more nucleic acids characteristic of the microorganism and analysis of the amplified one or more nucleic acids to identify the microorganism present in the blood sample; wherein the blood sample mixed with the differential lysis buffer has a pH of about 7.0 to 8.0, and the buffering agent has a useful pH buffering range of about 8.6 to 11.4.

[0069] B2. The method of clause B1, wherein prior to amplification, the assay further comprises lysis of the microorganism and recovery of nucleic acid from the microorganism, wherein the recovered nucleic acid is subjected to amplification for identification of the microorganism present in the blood sample.

[0070] B3. The method of any one of clauses B1 or B2, wherein the non-ionic surfactant is a polyoxyethylene (POE) ether.

[0071] B4. One or more of the methods of clauses B1-B3, wherein the non-ionic surfactant is selected from the group consisting of Triton X-114, NP-40, Arlasolve 200, BrijO10 (also known as Brij 96 / 97), octyl β-D-glucopyranoside, saponin, nonaethylene glycol monododecyl ether (C12E9, Polydosenol), and combinations thereof.

[0072] B5. One or more of the methods of clauses B1-B4, wherein the buffering substance is selected from the group consisting of CABS, CAPS, CAPSO, CHES, and combinations thereof.

[0073] B6. The method of any one or more of clauses B1-B5, wherein the buffering agent is CAPS, and CAPS has a pH buffering range of about 9.7-11.1 and a pKa of about 10.4 at 25° C.

[0074] B7. combining the blood sample with a differential lysis buffer in a centrifugal concentrator; combining the blood sample with a differential lysis buffer for a first time to obtain a lysate; centrifuging the centrifugal concentrator to pellet microorganisms from the blood sample disposed within the chamber; depressing the plunger to open the seal and cause the pellet to emerge from an opening at the second end of the chamber; wherein the centrifugal concentrator further comprises: a chamber having an open portion at a first end and a sealing portion at a second end, the sealing portion configured to seal the second open portion at the second end of the chamber; and a plunger movably disposed at least partially inside the chamber, the plunger designed to be actuated to open the seal; one or more of the methods set forth in clauses B1 to B6,

[0075] B8. The method of one or more of clauses B1-B7, wherein depressing the plunger, opening the seal portion, and producing a pellet includes depressing the plunger into sealing engagement with a portion of the body, and expelling the pellet from the second end by opening the seal under pressure.

[0076] B9. Optionally, forming the pellet in a cannulated vial comprising a vial body having an interior volume with a sample buffer disposed therein and a cannula extending from a bottom surface of the vial body; a cannula that does not extend into the vial body, the cannula having a first end adjacent a bottom surface of the vial body and a second end; the vial body further comprises a filter located near a bottom surface of the vial body designed to filter the body fluid prior to entry into the cannula, where the filter has a pore size of sufficient diameter to allow fungal, viral, protozoan, and / or bacterial organisms to pass therethrough and enter the cannula, but small enough to trap larger particulate matter; One or more of the methods set out in clauses B1 to B8

[0077] B10. The method of one or more of clauses B1-B9, further comprising disposing a second end of the cannula within a first port of a self-contained assay device, wherein the first port of the self-contained assay device is provided under vacuum to direct a volume of bodily fluid from the vial body, through the cannula, and into the self-contained assay device.

[0078] B11. A self-contained assay device comprising: a cell lysis zone fluidly connected to the first port and designed to lyse microorganisms; a nucleic acid preparation zone designed for purifying nucleic acids from microorganisms, fluidly connected to the cell lysis zone; a first-stage reaction zone fluidly connected to the nucleic acid preparation zone, the first-stage reaction zone including a first-stage reaction chamber designed for first-stage amplification of nucleic acid purified from the microorganism; and a second-stage reaction zone fluidly connected to the first-stage reaction zone, the second-stage reaction zone comprising a plurality of second-stage reaction chambers, each of which comprises a primer pair designed for further amplification of organism-specific nucleic acid purified from the microorganism, the second-stage reaction zone designed for simultaneous thermal cycling in all of the plurality of second-stage reaction chambers and for performing post-amplification nucleic acid melting and melting curve analysis to identify microorganisms present in the blood sample; One or more of the methods of clauses B1 to B10 further comprising:

[0079] B12. The method of one or more of clauses B1-B11, wherein the centrifugal concentrator and the vial body of the cannula processing vial are designed to engage with each other to couple the centrifugal concentrator with the cannula processing vial, and wherein the vial body of the cannula processing vial is designed to surround the second end and thus collect the pellets expressed from the second end of the chamber.

[0080] B13. The method of one or more of clauses B1-B12, wherein the second end of the centrifugal concentrator includes a first engagement portion and the vial body of the cannulated processing vial includes a second complementary engagement portion for fixedly coupling the centrifugal concentrator to the cannulated processing vial.

[0081] B14. The method of any one or more of clauses B1-B13, wherein the first mating portion and the second mating portion include threads for threadably coupling the centrifugal concentrator to a cannulated processing vial.

[0082] B15. The method of one or more of clauses B1-B14, further comprising: engaging the centrifugal concentrator and the cannulated processing vial to direct a volume of bodily fluid from the vial body through the cannula and into the self-contained assay device; removing the cannula of the cannulated processing vial from the first port of the self-contained assay device; and disposing of the centrifugal concentrator and the cannulated processing vial.

[0083] B16. The method of any one or more of clauses B1-B15, wherein the opening at the first end of the centrifugal concentrator includes a septum or the like designed for aseptically loading the sample mixed with the differential lysis buffer into the centrifugal concentrator.

[0084] B17. The method of one or more of clauses B1-B16, wherein the centrifugal concentrator does not include a high-density cushion.

[0085] B18. One or more of the methods of clauses B1 to B17, not including one or more of the following: pre-treating the blood sample, a blood culture step, a step of subculturing the blood sample to identify microorganisms present in the blood sample, or a DNase step to digest genomic DNA in the lysate.

[0086] B19. The method of any one or more of clauses B1-B18, wherein steps (a)-(c) are completed within a time range of about 10-20 minutes.

[0087] B20. The method of any one or more of clauses B1-B19, wherein steps (d) and (e) are completed within a time range of about 15-75 minutes.

[0088] B21. The method of any one or more of clauses B1-B20, wherein steps (a)-(e) are completed within a time range of about 25-95 minutes.

[0089] B22. The method of any one of clauses B1 to B21, wherein the first time for obtaining the lysate is within the range of about 2 to 10 minutes, preferably about 5 minutes.

[0090] B23. The method of any one or more of clauses B1 to B22, wherein obtaining the lysate does not include any additional steps other than combining.

[0091] C1. A blood sample known to contain or capable of containing a microorganism; and A differential lysis buffer is combined with the blood sample, the differential lysis buffer comprising an aqueous medium, a buffer substance, and a non-ionic surfactant. and having a pH of about 7.0 to 8.0, wherein the buffer has a useful pH buffering range of about 8.6 to 11.4 and a pKa within the range of about 9.5 to about 10.7 at 25° C.

[0092] C2. The composition of clause C1, wherein the nonionic surfactant is a polyoxyethylene (POE) ether.

[0093] C3. One or more compositions of clause C1 or C2, wherein the non-ionic surfactant is selected from the group consisting of Triton X-114, NP-40, Arlasolve 200, BrijO10 (also known as Brij 96 / 97), octyl β-D-glucopyranoside, saponin, nonaethylene glycol monododecyl ether (C12E9, Polydosenol), and combinations thereof.

[0094] C4. One or more compositions of clauses C1-C3, wherein the buffering agent is selected from the group consisting of CABS, CAPS, CAPSO, CHES, and combinations thereof.

[0095] C5. One or more compositions of clauses C1-C4, wherein the buffering agent is CAPS having a pH buffering range of about 9.7-11.1 and a pKa of about 10.4 at 25° C.

[0096] C6. One or more compositions of clauses C1-C5, wherein the buffer is substantially positively charged at a pH of about 7.0-8.0.

[0097] C7. Compositions according to any one or more of clauses C1 to C6, which are DNase-free

[0098] C8. A blood sample known to contain or capable of containing a microorganism; and Differential lysis buffer containing a buffer substance and a non-ionic detergent and having a pH of about 7.0 to 8.0, wherein the buffering agent is CAPS, which has a useful pH buffering range of about 9.7 to 11.1 and a pKa of about 10.4 at 25° C.

[0099] D1. A blood sample known to contain or capable of containing a microorganism; and Differential lysis buffer containing a buffer substance and a non-ionic detergent having a pH of about 7.0 to 8.0, wherein the buffering substance has a useful pH buffering range of about 8.6 to 11.4 and a pKa in the range of about 9.5 to about 10.7 at 25° C.; a composition comprising a lysate comprising lysed hemocytes and, if present, non-lysed microorganisms; 1. A centrifugal concentrator designed to pellet unlysed microorganisms in a lysate, comprising: a chamber having an open portion at a first end and a sealing portion at a second end, the sealing portion configured to seal the second open portion at the second end of the chamber; and a plunger movably disposed at least partially inside the chamber, the plunger designed to be actuated to open the seal; a centrifugal concentrator comprising: a cannulated processing vial designed to couple with the second end of the centrifugal concentrator for receiving the microbial pellet from the centrifugal concentrator, optionally, a vial body having an interior volume having a sample buffer therein, and a cannula extending from a bottom surface of the vial body, the cannula having a first end adjacent the bottom surface of the vial body and a second end not extending into the vial body; and The vial body further includes a filter located near a bottom surface of the vial body designed to filter the body fluid before it enters the cannula, where the filter has a pore size of sufficient diameter to allow fungal, viral, protozoan, and / or bacterial organisms to pass therethrough and enter the cannula, but small enough to trap larger particulate matter. Cannula processing vial containing A system that includes

[0100] D2. The system of clause D1, wherein the centrifugal concentrator does not include a high density cushion.

[0101] D3. The system of one or more of clauses D1 and D2, further comprising a self-contained assay device having a first port designed to receive a second end of a cannula for introduction of a sample into the self-contained assay device, wherein the first port of the self-contained assay device is provided under vacuum to direct a volume of sample from the vial body, through the cannula, and into the self-contained assay device.

[0102] D4. A self-contained assay device, a cell lysis zone fluidly connected to the first port, the cell lysis zone designed to lyse a microorganism; a nucleic acid preparation zone fluidly connected to the cell lysis zone, the nucleic acid preparation zone designed for purifying nucleic acids from the microorganism; a first-stage reaction zone fluidly connected to the nucleic acid preparation zone, the first-stage reaction zone comprising a first-stage reaction chamber designed for first-stage amplification of nucleic acids purified from a microorganism; and a second-stage reaction zone fluidly connected to the first-stage reaction zone, the second-stage reaction zone comprising a plurality of second-stage reaction chambers, each of the second-stage reaction chambers comprising a primer pair designed for further amplification of organism-specific nucleic acid purified from the microorganism, the second-stage reaction zone designed for simultaneous thermal cycling in all of the plurality of second-stage reaction chambers and for performing post-amplification nucleic acid melting and melting curve analysis to identify microorganisms present in the blood sample; One or more systems of clauses D1 to D3 further comprising:

[0103] D5. One or more systems of clauses D1-D4, wherein the centrifugal concentrator and the vial body of the cannula processing vial are designed to engage with each other to couple the centrifugal concentrator with the cannula processing vial, and wherein the vial body of the cannula processing vial is designed to surround the second end, and thus the vial body of the cannula processing vial is designed to collect pellets expressed from the second end of the chamber.

[0104] D6. The system of one or more of clauses D1-D5, wherein the second end of the centrifugal concentrator includes a first engagement portion and the vial body of the cannulated processing vial includes a complementary second engagement portion for fixedly coupling the centrifugal concentrator to the cannulated processing vial.

[0105] D7. The system of any one or more of clauses D1-D6, wherein the first engaging portion and the second engaging portion include threads for threadably coupling the centrifugal concentrator to a cannulated processing vial.

[0106] D8. One or more systems of clauses D1-D7, wherein the opening at the first end of the centrifugal concentrator includes a septum or the like designed for aseptically filling the centrifugal concentrator with a sample mixed with a differential lysis buffer.

[0107] D9. A system of one or more of clauses D1-D8, wherein the centrifugal concentrator does not include a high-density cushion

[0108] D10. One or more systems of clauses D1 to D9, wherein the non-ionic surfactant of the differential lysis buffer is a polyoxyethylene (POE) ether.

[0109] D11. One or more systems of clauses D1-D10, wherein the non-ionic detergent of the differential lysis buffer is selected from the group consisting of Triton X-114, NP-40, Arlasolve 200, BrijO10 (also known as Brij96 / 97), octyl β-D-glucopyranoside, saponin, nonaethylene glycol monododecyl ether (C12E9, polydosenol), and combinations thereof.

[0110] D12. One or more systems of clauses D1-D11, wherein the buffer substance of the differential lysis buffer is selected from the group consisting of CABS, CAPS, CAPSO, CHES, and combinations thereof.

[0111] D13. One or more systems of clauses D1-D12, wherein the buffer substance of the differential lysis buffer is CAPS, and CAPS has a useful pH buffering range of about 9.7-11.1 and a pKa of about 10.4 at 25° C.

[0112] D14. One or more systems of clauses D1 to D13, wherein the buffer substance of the differential lysis buffer is substantially positively charged at a pH of about 7.0 to 8.0.

[0113] D15. One or more systems of clauses D1 to D14, wherein the differential lysis buffer is DNase-free.

[0114] E1. A method for isolating and identifying microorganisms, comprising: (a) providing a blood sample known to contain or capable of containing a microorganism; (b) mixing the blood sample with a differential lysis buffer having a pH to obtain a lysate containing lysed hemocytes and unlysed microorganisms; (c) placing the blood sample mixed with the differential lysis buffer into a centrifugal concentrator; (d) centrifuging the centrifugal concentrator to pellet microorganisms from the blood sample disposed within the chamber; (e) adding the microorganism to a self-contained assay device that contains one or more reagents required to identify the microorganism; (f) identifying microorganisms present in the blood sample; and wherein adding the microorganism to the self-contained assay device comprises depressing a plunger, opening a sealed portion, and causing a pellet to form from an opening at the second end of the chamber, and wherein identifying comprises isolating one or more nucleic acids characteristic of the microorganism from the microorganism, performing nucleic acid amplification, and performing post-amplification nucleic acid melting and melting curve analysis to identify the microorganism present in the blood sample; Here, the centrifugal concentrator a chamber having an open portion at a first end and a sealing portion at a second end, the sealing portion configured to seal the second open portion at the second end of the chamber; and a plunger movably disposed at least partially inside the chamber, the plunger designed to be actuated to open the seal; A method comprising:

[0115] E2. The self-contained assay device comprises: a first port provided under vacuum to direct a quantity of the pellet into a self-contained assay device; a cell lysis zone fluidly connected to the first port, the cell lysis zone designed to lyse a microorganism; a nucleic acid preparation zone fluidly connected to the cell lysis zone, the nucleic acid preparation zone designed for purifying nucleic acids from the microorganism; a first-stage reaction zone fluidly connected to the nucleic acid preparation zone, the first-stage reaction zone comprising first-stage reaction chambers designed for performing a first-stage multiplex amplification on one or more nucleic acids; a second-stage reaction zone fluidly connected to the first-stage reaction zone, the second-stage reaction zone comprising a plurality of second-stage reaction chambers, each of the second-stage reaction chambers comprising a primer pair designed for further amplification of a specific nucleic acid purified from one of the microorganisms, the second-stage reaction zone designed for simultaneous thermal cycling in all of the plurality of second-stage reaction chambers. and performing a first-stage multiplex amplification in a first-stage reaction zone to obtain first-stage amplification products; diluting the first-stage amplification products; separating the diluted first-stage amplification products in a plurality of second-stage reaction chambers; performing a second-stage amplification in the second-stage amplification chambers; and performing a post-amplification nucleic acid melt and a post-second-stage amplification melting curve analysis to identify microorganisms present in the blood sample. The method of clause E1, further comprising:

[0116] E3. The method of one or more of clauses E1 and E2, wherein depressing the plunger, opening the seal portion, and producing a pellet includes depressing the plunger into sealing engagement with a portion of the body, and expelling the pellet from the second end under pressure by opening the seal.

[0117] E4. optionally forming a pellet in a cannulated vial having a sample buffer therein, the cannulated vial including a vial body having an interior volume and a cannula extending from a bottom surface of the vial body; a cannula that does not extend into the vial body, the cannula having a first end adjacent a bottom surface of the vial body and a second end; the vial body further comprises a filter located near a bottom surface of the vial body designed to filter the body fluid before it enters the cannula, where the filter has a pore size of sufficient diameter to allow fungal, viral, protozoan, and / or bacterial organisms to pass therethrough and enter the cannula, but small enough to trap larger particulate matter; One or more of the methods described in clauses E1 to E3

[0118] E5. The method of one or more of clauses E1-E4, further comprising disposing a second end of the cannula within a first port of a self-contained assay device, wherein the first port of the self-contained assay device is provided under vacuum to direct a volume of bodily fluid from the vial body, through the cannula, and into the self-contained assay device.

[0119] E6. The method of any one or more of clauses E1-E5, wherein steps (b)-(d) are performed in a single tube.

[0120] E7. The method of any one or more of clauses E1-E6, wherein the differential lysis buffer is a single buffer provided in a single tube.

[0121] E8. The method of one or more of clauses E1-E7, wherein the differential lysis buffer does not contain DNase or protease and the method does not include the step of adding exogenous DNase or protease to a single tube.

[0122] E9. One or more of the methods of clauses E1-E8, not including one or more of the following: pre-treating the blood sample, a blood culture step, a step of subculturing the blood sample to identify microorganisms present in the blood sample, or a DNase step to digest genomic DNA in the lysate.

[0123] E10. The method of any one or more of clauses E1-E9, wherein steps (a)-(d) are completed within a time range of about 10-20 minutes.

[0124] E11. The method of any one or more of clauses E1-E10, wherein steps (e) and (f) are completed within a time range of about 15-75 minutes.

[0125] E12. One or more of the methods of clauses E1-E11, wherein steps (a)-(f) are completed within a time range of about 25-95 minutes.

[0126] This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as a contribution in determining the scope of the claimed subject matter.

[0127] Additional features and advantages will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by the practice of the invention. The features and advantages may be realized and obtained by means of the instruments and combinations particularly pointed out in the appended claims. These and other features will become more fully apparent from the following description and the appended claims, or may be learned by the practice of the invention as set forth hereinafter. [Brief description of the drawings]

[0128] [Figure 1] 1 shows a movable pouch useful for self-contained PCR. [Diagram 2] 2 is an exploded perspective view of an apparatus including the pouch of FIG. 1 and for use with the pouch of FIG. 1. [Diagram 3] The pouch of FIG. 1 is shown with the bladder component of FIG. [Figure 4] 3 illustrates a motor used in an exemplary embodiment of the device of FIG. 2. [Diagram 5] 1 is a schematic diagram of one embodiment of a differential lysis and centrifugation method using the systems and devices described herein. [Figure 6A] FIG. 2 is an isometric view of a centrifugal concentrator in accordance with one embodiment of the present invention. [Figure 6B]FIG. 6B is a side view of the centrifugal concentrator of FIG. 6A. [Figure 6C] 6C shows the same view of the centrifugal concentrator as in FIG. 6B with the cap removed. [Figure 6D] FIG. 2 is another isometric view of the centrifugal concentrator. [Figure 6E] FIG. 2 is a detailed view of one end of the centrifugal concentrator. [Figure 6F] FIG. 6F is a cross-sectional view of the distal end of the centrifugal concentrator shown in FIG. 6E. [Figure 6G] FIG. 6B is an isometric view of the plunger of the centrifugal concentrator of FIGS. 6A-6F. [Figure 7] 1 is an example of a workflow using differential lysis buffer. [Figure 8] 1 is a bar graph comparing Differential Lysis Buffer with several other protocols. [Figure 9] 1 is a bar graph illustrating cell recovery with differential lysis buffer. [Figure 10] 1 illustrates the effectiveness of removing human genomic DNA for an exemplary differential lysis and centrifugation method. [Figure 11] Data showing that the Differential Lysis Buffer can selectively lyse eukaryotic host cells while leaving microbial cells intact. [Figure 12] Illustrates the workflow for recovery and detection efficiency of microorganisms from whole blood. [Figure 13] 13 illustrates the mean recovery of microorganisms in the testing workflow illustrated in FIG. 12. [Figure 14] 13 illustrates the average inoculum and recovery of microorganisms in the testing workflow illustrated in FIG. [Figure 15A-C] FIG. 1 illustrates a flow-through method for animal cell lysis, culture, and concentration of microorganisms. [Figure 16] Illustrates a filtration method for the isolation and concentration of microorganisms. [Figure 17A-C] 1A-1D illustrate schematics of different filtration structures that can be used to separate cells by size. [Figure 18] 18A-18C show schematics of different types of pillar filters, such as (18A) polygonal, (18B) U-shaped, and (18C) butterfly-shaped micropillar geometries. [Figure 19] FIG. 1 illustrates a schematic of the separation of large and small cells within a structure with a series of micropillars and cross-flow of buffer and cell suspension. [Figure 20] Schematic depiction of enrichment of large and small cells by migration along an ovoid filter unit. [Figure 21] 1 is a graph of absorbance versus incubation time illustrating the lysis of blood samples over time with various differential lysis buffer formulations. [Figure 22] 1 is a bar graph illustrating increasing organism concentration for several types of organisms and blood anticoagulants. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0129] Examples of embodiments are described below with reference to the accompanying drawings. Since many configurations and embodiments are possible without departing from the spirit and teachings of the present disclosure, the present disclosure should not be construed as being limited to the example embodiments described herein. Rather, these example embodiments are provided so that this disclosure will be thorough and complete, and will convey the scope of the disclosure to those skilled in the art. In the drawings, the sizes and relative sizes of layers and regions may be exaggerated for clarity. Like reference numbers refer to like elements throughout the description.

[0130] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure pertains. It will be further understood that terms as defined in commonly used dictionaries should be interpreted to have a meaning consistent with their meaning in connection with this application and related art, and should not be interpreted in an idealized or overly formal sense unless specifically and explicitly defined herein. The terms used in the description of the invention herein are merely intended to describe certain embodiments and are not intended to limit the invention. Although several methods and materials similar or equivalent to those described herein can be used in the practice of this disclosure, only certain representative materials and methods are described herein.

[0131] All publications, patent applications, patents, or other references mentioned herein are incorporated by reference in their entirety. In the event of a conflict in terminology usage, the present specification shall control.

[0132] Various aspects of the present disclosure, including devices, systems, methods, and the like, may be illustrated in connection with one or more representative implementations. As used herein, the terms "representative" and "exemplary" mean "serving as an example, instance, or illustration," and should not necessarily be construed as preferred or advantageous over other implementations disclosed herein. Moreover, references to "implementations" or "embodiments" of the present disclosure or invention include specific references to one or more embodiments thereof, and vice versa, and are intended to provide illustration without limiting the scope of the invention, which is indicated by the appended claims rather than the following description.

[0133] As used herein and in the appended claims, the singular forms "a," "an," and "the" will be recognized as including plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a tile" will include one, two, or more tiles. Similarly, references to plural referents should be construed as including a single referent and / or multiple referents unless the context indicates or clearly dictates otherwise. Thus, reference to "tiles" does not necessarily require a plurality of such tiles. Instead, it will be understood that one or more tiles are contemplated herein apart from combinations.

[0134] As used throughout this application, the terms "can" and "may" are used in a permissive sense (i.e., meaning having the ability to) rather than a mandatory sense (i.e., meaning must). In addition, the terms "including," "having," "involving," "containing," "characterized by," variations thereof (e.g., "includes," "has," "involves," "contains," etc.), and similar terms, when used herein, including the claims, are inclusive and / or open-ended and are intended to have the same meaning as the term "comprising" and variations thereof (e.g., "comprise" and "comprises") and, illustratively, do not exclude additional, unrecited elements or method steps.

[0135] As used herein, directional and / or subjective terms such as "top," "bottom," "left," "right," "upper," "lower," "upper," "lower," "inner," "outer," "internal," "external," "interior," "exterior," "proximal," "distal," "forward," "reverse," and the like, may be used alone to indicate relative directions and / or orientations and may not be intended to specifically limit the scope of the present disclosure, including the specification, invention, and / or claims.

[0136] When an element is referred to as being "coupled," "connected," or "responsive" to another element, or "to" another element, it may be directly coupled, connected, or responsive to or to the other element, or intervening elements may be present. In contrast, when an element is referred to as being "directly coupled," "directly connected," or "directly responsive" to another element, or "directly to" another element, it will be understood that there are no intervening elements present.

[0137] Example embodiments of the inventive concepts are described herein with reference to cross-sectional illustrations that are schematic illustrations of idealized embodiments (and intermediate-stage structures) of the example embodiments. As such, variations from the shapes of the illustrations as a result, for example, of manufacturing techniques and / or tolerances, are to be expected. Thus, the example embodiments of the inventive concepts should not be construed as limited to the particular shapes of regions illustrated herein, but will include, for example, deviations in shapes that result from manufacturing. Thus, the regions illustrated in the drawings are schematic in nature and their shapes are not intended to illustrate the actual shape of a region of a device and are not intended to limit the scope of the example embodiments.

[0138] Terms such as "first," "second," and the like may be used herein to describe various elements, but it will be understood that these elements should not be limited by these terms. These terms are merely used to distinguish one element from another. Thus, a "first" element could be termed a "second" element without departing from the teachings of the present embodiments.

[0139] It is also understood that the various implementations described herein can be utilized in combination with any other implementations described or disclosed without departing from the scope of the present disclosure. Thus, products, members, elements, devices, apparatus, systems, methods, processes, compositions, and / or kits according to a particular implementation of the present disclosure may include, encompass, or otherwise comprise properties, features, components, members, elements, steps, etc. described in other implementations (including systems, methods, apparatus, etc.) disclosed herein without departing from the scope of the present disclosure. Thus, specific features associated with one implementation should not be construed as being limited to application only within that implementation.

[0140] The headings used herein are for organizational purposes only and are not meant to be used to limit the scope of the description or the claims. To facilitate understanding, where possible, similar reference numbers have been used to designate similar elements common to the figures. Furthermore, where possible, similar element numbering has been used in the various figures. Additionally, alternative designs of a particular element may each include a different letter appended to the element number.

[0141] The term "about" is used herein to mean roughly, or approximately within a range. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the upper and lower boundaries of the numerical values ​​set forth. In general, the term "about" is used herein to modify numerical values ​​above and below a variance of 5% of the stated value. When such a range is expressed, another embodiment includes from one particular value and / or to the other particular value. Similarly, it will be understood that particular values ​​form another embodiment when the values ​​are expressed as approximations by use of "about" preceding them. It will further be understood that each endpoint of the range is significant in any event both in relation to the other endpoint and independently of the other endpoint.

[0142] The term "or" as used herein means any one member of a particular list and also includes any combination of members of that list.

[0143] As used herein, the term "microorganism" is intended to encompass organisms, generally unicellular, that can be propagated and handled in a laboratory, including, but not limited to, gram-positive or gram-negative bacteria, yeasts, molds, and parasites. Non-limiting examples of gram-negative bacteria of the present invention include bacteria of the following genera: Pseudomonas, Escherichia, Salmonella, Shigella, Enterobacter, Klebsiella, Serratia, Proteus, Campylobacter, Haemophilus, Morganella, Vibrio, Yersinia, Acinetobacter, Stenotrophomonas, Brevundimonas, and Brevundimonas. evundimonas, Ralstonia, Achromobacter, Fusobacterium, Prevotella, Branhamella, Neisseria, Burkholderia, Citrobacter, Hafnia, Edwardsiella, Aeromonas, Moraxella, Brucella, Pasteurella, Providencia, and Legionella.Non-limiting examples of Gram-positive bacteria of the present invention include bacteria of the following genera: Enterococcus, Streptococcus, Staphylococcus, Bacillus, Paenibacillus, Lactobacillus, Listeria, Peptostreptococcus, Propionibacterium, Clostridium, Bacteroides, Gardnerella, Kocuria, Lactococcus, Leuconostoc, Micrococcus, Mycobacteria, and Corynebacteria. Non-limiting examples of yeasts and molds of the present invention include yeasts and molds of the following genera: Candida, Cryptococcus, Nocardia, Penicillium, Alternaria, Rhodotorula, Aspergillus, Fusarium, Saccharomyces, and Trichosporon. Non-limiting examples of parasites of the present invention include parasites of the following genera: Trypanosoma, Babesia, Leishmania, Plasmodium, Wucheria, Brugia, Onchocerca, and Naegleria.

[0144] In one aspect, as described in further detail herein, microorganisms from a sample or growth medium can be separated and examined to characterize and / or identify the microorganisms present in the sample. As used herein, the term "separate" is intended to encompass any sample of microorganisms that have been removed, concentrated, or otherwise separated from its original state or from a growth medium or culture medium. For example, in accordance with the present invention, microorganisms may be separated (e.g., as separate samples) from non-microorganisms or non-microbial components that would otherwise interfere with the characterization and / or identification. The term may include microorganisms that have been separated from a mixture by centrifugation, filtration, or any other separation technique known in the art. As such, a separated microbial sample may include a collection of microorganisms and / or components thereof that are concentrated from or otherwise separated from the original sample, and may range from a tightly packed, dense clump of microorganisms to a diffuse layer of microorganisms. Non-microbial components separated from microorganisms may include non-microbial cells (e.g., blood cells and / or other tissue cells) and / or any components thereof. In one aspect, the microorganisms are separated from a lysate mixture comprising lysed non-microbial cells and substantially intact microbial cells.

[0145] In some embodiments, the separation of the sample of microorganisms from its original state or from the growth or medium is incomplete. In other words, the sample of microorganisms is not completely separated from other components of the sample or from the growth or medium due to removal, concentration, or otherwise setting away from its original state of the microorganisms. In some cases, there is a minimal amount of debris from the sample or from the growth or medium. For example, the amount of debris or growth or medium present in the separated sample may be insufficient to interfere with the identification or characterization of the microorganism or further growth of the microorganism. In some embodiments, the separated sample is 99% pure from contaminating components, but it may also be 95% pure, 90% pure, 80% pure, 70% pure, 60% pure, 50% pure, or a minimum purity that still allows identification of the microorganism in the separated sample by downstream identification techniques.

[0146] In yet another aspect described in more detail herein, microorganisms from a sample or growth medium can be pelleted and examined to characterize and / or identify the microorganisms present in the sample. As used herein, the term "pellet" is intended to encompass any sample of microorganisms that have been compacted or accumulated into a mass of microorganisms. For example, microorganisms from a sample can be compacted or accumulated into a mass at the bottom of a tube by centrifugation or other methods known in the art. The term includes the collection of microorganisms (and / or components thereof) on the bottom and / or sides of a vessel after centrifugation. In accordance with the present invention, microorganisms can be pelleted (e.g., as a substantially purified microbial pellet) from non-microbial or non-microbial components that would normally interfere with characterization and / or identification.

[0147] The term "nucleic acid" as used herein refers to naturally occurring or synthetic oligonucleotides or polynucleotides, either single-stranded or double-stranded, sense or antisense, DNA or RNA or DNA-RNA hybrids, capable of hybridizing with a complementary nucleic acid by Watson-Crick base pairing. Nucleic acids of the invention may also include nucleotide analogs (e.g., BrdU), and non-phosphodiester internucleoside linkages (e.g., peptide nucleic acid (PNA) or thiodiester linkages). In particular, nucleic acids may include, but are not limited to, DNA, RNA, mRNA, rRNA, cDNA, gDNA, ssDNA, dsDNA, or any combination thereof.

[0148] "Probe", "primer" or "oligonucleotide" refers to a single-stranded nucleic acid molecule of defined sequence that can base-pair to a second nucleic acid molecule ("target") that contains a complementary sequence. The stability of the resulting hybrid depends on the length, GC content, and extent of base-pairing that occurs. The extent of base-pairing is affected by parameters such as the degree of complementarity between the probe and target molecule and the degree of stringency of the hybridization conditions. The degree of hybridization stringency is affected by parameters such as temperature, salt concentration, and the concentration of organic molecules such as formamide, and is determined by methods known to those of skill in the art. Probes, primers, and oligonucleotides may be detectably labeled, either radioactively, fluorescently, or non-radioactively, by methods well known to those of skill in the art. dsDNA binding dyes may be used to detect dsDNA. It is understood that while "primers" are specifically designed to be extended by a polymerase, "probes" or "oligonucleotides" may or may not be so designed.

[0149] "dsDNA binding dye" refers to a dye that fluoresces differently, usually by fluorescing more strongly, when bound to double-stranded DNA than when bound to single-stranded DNA or free in solution. When referring to dsDNA binding dyes, it is understood that any suitable dye may be used herein, and some non-limiting exemplary dyes are described in U.S. Pat. No. 7,387,887, which is incorporated herein by reference. As known in the art, other signal-generating substances, such as enzymes, antibodies, etc., may be used to detect nucleic acid amplification and melting.

[0150] "Specifically hybridize" means that a probe, primer, or oligonucleotide recognizes and physically interacts (i.e., base pairs) with a substantially complementary nucleic acid (e.g., a sample nucleic acid) under high stringency conditions, and does not substantially base pair with other nucleic acids.

[0151] "High stringency conditions" typically refer to conditions performed at about melting temperature (Tm)-5°C (i.e., 5° below the Tm of the probe). Functionally, high stringency conditions are used to identify nucleic acid sequences with at least 80% sequence identity.

[0152] "Lysing particles" refers to various particles or beads for lysis of cells, viruses, spores, and other materials that may be present in a sample. In various examples, zirconium ("Zr") silicate or ceramic beads are used, but other lysis particles are known and are included within the scope of the term, including glass and sand lysis particles. The term "cell lysis components" may include lysis particles as known in the art, but may also include other components, such as components for chemical lysis.

[0153] While PCR is the amplification method used in the examples herein, it is understood that any amplification method using primers may be suitable. Such suitable methods include polymerase chain reaction (PCR); strand displacement amplification (SDA); nucleic acid sequence-based amplification (NASBA); cascade rolling circle amplification (CRCA), loop-mediated isothermal amplification of DNA (LAMP); isothermal and chimeric primer-initiated amplification of nucleic acid (ICAN); target-based helicase-dependent amplification (HDA); transcription-mediated amplification (TMA), etc. Therefore, when the term PCR is used, it should be understood to include other alternative amplification methods. In amplification methods without separate cycles, reaction time may be used when measurements are made in cycles, doubling times, or crossing points (Cp), and additional reaction time may be added when additional PCR cycles are added in the embodiments described herein. Therefore, it is understood that protocols may need to be adjusted.

[0154] While various examples herein refer to human targets and human pathogens, these examples are merely illustrative, and the methods, kits, and devices described herein can be used to detect or sequence a wide variety of nucleic acid sequences from a wide variety of samples, including human, animal, industrial, and environmental.

[0155] In various embodiments disclosed herein, illustratively, a self-contained nucleic acid analysis pouch is used to assay samples for the presence of various biological substances, illustratively antigens and nucleic acid sequences, within a single closed system. Such systems, including pouches and devices for use with pouches, are disclosed in more detail in U.S. Pat. Nos. 8,394,608; and 8,895,295; and 10,464,060, which are incorporated herein by reference. However, it is understood that such pouches are merely illustrative and that the nucleic acid preparation and amplification reactions discussed herein may be performed in any of a variety of open or closed system sample vessels as known in the art, including 96-well plates, plates of other shapes, arrays, carousels, and the like, using a variety of nucleic acid purification and amplification systems as known in the art. While terms such as "sample wells," "amplification wells," and "amplification vessels" are used herein, these terms are meant to encompass wells, tubes, and various other reaction vessels as used in these amplification systems. In one embodiment, the pouch is used to assay for multiple pathogens. The pouch may illustratively include one or more blisters used as sample wells in a closed system. Illustratively, various steps may be performed in the disposable pouch, including nucleic acid preparation, primary high-volume multiplex PCR, dilution of the primary amplification product, and secondary PCR, optionally ending with post-amplification analysis such as real-time detection or melting curve analysis. Furthermore, while various steps may be performed in the pouch of the present invention, it is understood that one or more of the steps may be omitted for a particular use, and the configuration of the pouch may be modified accordingly. While in many embodiments herein a multiplex reaction is used in the first-stage amplification, it is understood that this is merely illustrative, and that in some embodiments the first-stage amplification may be singleplex. In one illustrative example, the first-stage singleplex amplification targets a housekeeping gene, and the second-stage amplification uses the difference in the housekeeping gene for identification.Thus, while in various embodiments a first-stage multiplex amplification is contemplated, it is understood that this is merely exemplary.

[0156] FIG. 1 shows an exemplary pouch 510 that may be used or redesigned in various embodiments. Pouch 510 is similar to FIG. 15 of U.S. Pat. No. 8,895,295, with similar items numbered similarly. Fitment 590 is provided with entry channels 515a-515l, which also serve as reagent or waste reservoirs. Illustratively, reagents may be lyophilized within fitment 590 and rehydrated prior to use. Blisters 522, 544, 546, 548, 564, and 566, along with their respective channels 514, 538, 543, 552, 553, 562, and 565, are similar to the like-numbered blisters of FIG. 15 of U.S. Pat. No. 8,895,295. The second-stage reaction zone 580 of FIG. 1 is similar to that of U.S. Pat. No. 8,895,295, although the second-stage wells 582 of the high density array 581 are arranged in a slightly different pattern. The more circular pattern of the high density array 581 of FIG. 1 may result in more uniform filling of the second-stage wells 582 with the edge wells removed. As shown, the high density array 581 is provided with 102 second-stage wells 582. The pouch 510 is suitable for use in a FilmArray® instrument (BioFire Diagnostics, LLC, Salt Lake City, UT). However, it is understood that the pouch embodiment is merely exemplary.

[0157] While other containers may be used, illustratively the pouch 510 may be formed of two layers of flexible plastic film or other flexible material, such as polyester, polyethylene terephthalate (PET), polycarbonate, polypropylene, polymethylmethacrylate, mixtures, combinations, and layers thereof, which may be made by any process known in the art, including extrusion, plasma deposition, and lamination. For example, each layer may be composed of one or more layers of a single type or two or more types of material laminated together. Metal foil or plastic with aluminum lamination may also be used. Other barrier materials are known in the art and can be sealed together to form blisters and channels. If a plastic film is used, the layers may be bonded together, illustratively by heat sealing. Illustratively, the material has low nucleic acid binding and low protein binding capacity.

[0158] For embodiments using fluorescence monitoring, plastic films with sufficiently low absorbance and autofluorescence at the effective wavelength are preferred. Such materials can be identified by testing different plastics, different plasticizers, and composite ratios, as well as different thicknesses of the film. For plastics with aluminum or other foil lamination, the portion of the pouch that will be read by the fluorescence detection device can be left without foil. For example, if fluorescence is monitored in second-stage well 582 of second-stage reaction zone 580 of pouch 510, one or both layers in well 582 will be left without foil. In a PCR example, a film laminate of polyester (Mylar, DuPont, Wilmington DE) with a thickness of about 0.0048 inches (0.1219 mm) and polypropylene film with a thickness of 0.001 to 0.003 inches (0.025 to 0.076 mm) works well. Illustratively, pouch 510 may be made from a transparent material capable of transmitting about 80% to 90% of incident light.

[0159] In an exemplary embodiment, the material is moved between the blisters by application of pressure, illustratively air pressure, to the blisters and channels. Thus, in embodiments using pressure, the pouch material is illustratively sufficiently flexible to allow the pressure to have the desired effect. The term "flexible" is used herein to describe the physical characteristics of the pouch material. The term "flexible" is defined herein as being easily deformable by the level of pressure used herein without cracking, breaking, craze, etc. For example, thin plastic sheets such as Saran™ wrap and Ziploc® bags, and thin metal foils such as aluminum foil, are flexible. However, flexibility is only required in certain areas of the blisters and channels, even in embodiments using air pressure. Furthermore, flexibility is only required on one side of the blisters and channels, so long as the blisters and channels are easily deformable. Other areas of the pouch 510 may be made of a rigid material or may be reinforced with a rigid material. Thus, when terms such as "flexible pouch" or "flexible sample container" are used, it is understood that flexibility is only required in some portions of the pouch or sample container.

[0160] Exemplarily, a plastic film may be used for the pouch 510. A sheet of metal, illustratively aluminum, or other suitable material may be rolled or otherwise cut to create a mold with a pattern of raised surfaces. When fitted with an air press (exemplarily A-5302-PDS, Janesville Tool Inc., Milton WI) and conditioned at an effective temperature, illustratively 195° C., the air press operates like a printing press, melting the sealing surface of the plastic film only where the mold contacts the film. Similarly, the plastic film used for the pouch 510 may be cut and welded together using a laser cutting and welding device. Various components such as PCR primers, antigen binding substrates, magnetic beads, and zirconium silicate beads (exemplarily spotted and dried on the film) may be sealed inside the various blisters as the pouch 510 is formed. Reagents for sample processing may be spotted onto the film, either together or separately, prior to sealing. In one embodiment, nucleotide triphosphates (NTPs) are spotted onto the film separately from the polymerase and primers, essentially eliminating the activity of the polymerase until the reaction can be hydrated with an aqueous sample. If the aqueous sample is heated prior to hydration, this creates the conditions for true hot start PCR, reducing or eliminating the need for expensive chemical hot start components. In another embodiment, the components may be provided in powder or pill form and placed into the blister before final sealing.

[0161] The pouch 510 may be used in a manner similar to that described in U.S. Patent No. 8,895,295. In one exemplary implementation, a 300 μl mixture containing a sample to be tested (100 μl) and a lysis buffer (200 μl) may be injected into an injection port (not shown) in the fitment 590 adjacent to the entry channel 515a, and the sample mixture may be directed into the entry channel 515a. Water may also be injected into a second injection port (not shown) in the entry channel 515l adjacent to the fitment 590, and the water may be distributed through channels (not shown) provided in the fitment 590, thereby hydrating up to eleven different reagents, each of which was previously provided in dry form in the entry channels 515b-515l. Exemplary methods and devices for injecting the sample and hydration liquid (e.g., water or buffer) are disclosed in U.S. Patent Publication No. 2014-0283945, which is incorporated herein by reference in its entirety, although it is understood that these methods and devices are merely exemplary and that other methods of introducing the sample and hydration liquid into the pouch 510 are within the scope of the present disclosure. These reagents may illustratively include lyophilized PCR reagents, DNA extraction reagents, wash solutions, immunoassay reagents, or other chemical entities. Illustratively, the reagents are for nucleic acid extraction, first-stage multiplex PCR, dilution of the multiplex reaction, and preparation of second-stage PCR reagents, as well as control reactions. In the embodiment shown in FIG. 1, all that needs to be injected is the sample solution in one injection port and water in the other injection port. After injection, the two injection ports may be sealed. For more information regarding the various configurations of the pouch 510 and the fitment 590, see U.S. Patent No. 8,895,295, previously incorporated by reference.

[0162] After injection, the sample may be transferred from injection channel 515a through channel 514 to lysis blister 522. Lysis blister 522 is provided with beads or particles 534, such as ceramic beads or other abrasive elements, and is designed for vortexing via impaction using rotating blades or paddles provided in the FilmArray® device. Bead milling by shaking, vortexing, sonication, and similar treatment of the sample in the presence of lysis particles, such as zirconium silicate (ZS) beads 534, is an effective method for forming a lysate. As used herein, it is understood that terms such as "lyse," "lysing," and "lysate" are not limited to disrupting cells, but include the disruption of non-cellular particles such as viruses. In another embodiment, a paddle beater using reciprocating or alternating paddles, as described, for example, in U.S. Patent Application Publication No. 2019-0344269, which is incorporated herein by reference in its entirety, may be used for lysis in this embodiment, as well as other embodiments described herein.

[0163] FIG. 4 shows a bead beating motor 819 including a blade 821 that can be mounted on the first side 811 of the support member 802 of the device 800 shown in FIG. 2. The blade can extend through the slot 804 and contact the pouch 510. However, it is understood that the motor 819 can be mounted on other structures of the device 800. In one exemplary embodiment, the motor 819 is a Mabuchi Motor RC-280SA-2865 DC motor (Chiba, Japan) mounted on the support member 802. In one exemplary embodiment, the motor rotates at 5,000-25,000 rpm, more illustratively 10,000-20,000 rpm, and even more illustratively about 15,000-18,000 rpm. For the Mabuchi Motor motor, 7.2V has been found to provide sufficient rpm for lysis. However, it is understood that the actual speed may be somewhat slower when the blade 821 is impacting the pouch 510. Other potentials and speeds may be used for lysis, depending on the motor and paddles used. Optionally, a controlled small amount of air may be provided to the lysis blister 522 adjacent to the bladder 822. In some embodiments, it has been found that partially filling one or more small amounts of air into an adjacent bladder aids in positioning and supporting the lysis blister during the lysis process. Alternatively, another structure, illustratively a rigid or compliant gasket or other retention structure around the lysis blister 522, may be used to confine the pouch 510 during lysis. It is also understood that the motor 819 is merely illustrative and that other devices may be used for milling, shaking, or vortexing the sample. In some embodiments, chemicals or heat may be used in addition to or instead of mechanical lysis.

[0164] Once the sample material has been sufficiently dissolved, the sample is transferred to a nucleic acid extraction zone, illustratively through channel 538, blister 544, and channel 543 to blister 546, where the sample is mixed with a nucleic acid binding substance, such as silica-coated magnetic beads 533. Alternatively, the magnetic beads 533 may be rehydrated, illustratively with liquid provided from one of entry channels 515c-515e, and then transferred through channel 543 to blister 544 and then through channel 538 to blister 522. The mixture may be incubated for a suitable time, illustratively from about 10 seconds to about 10 minutes. A retractable magnet located inside blister 546 adjacent the device captures the magnetic beads 533 from the solution and pellets them against the interior surface of blister 546. If incubation is performed in blister 522, multiple portions of the solution may need to be transferred to blister 546 for capture. Liquid is then transferred out of blister 546, through blister 544, and back to blister 522, where it is used as a waste container. One or more wash buffers from one or more of injection channels 515c-515e are provided to blister 546 via blister 544 and channel 543. Optionally, the magnet is retracted and magnetic beads 533 are washed by moving the beads back and forth from blisters 544 and 546 through channel 543. Once magnetic beads 533 have been washed, they are recaptured in blister 546 by activation of the magnet, and then the wash solution is transferred to blister 522. This process may be repeated as necessary to wash lysis buffer and sample debris from nucleic acid-bound magnetic beads 533.

[0165] After washing, elution buffer stored in injection channel 515f is transferred to blister 548 and the magnet is retracted. Solution is circulated between blisters 546 and 548 via channel 552, disrupting the pellet of magnetic beads 533 in blister 546 and allowing the captured nucleic acid to dissociate from the beads and go into solution. When the magnet is reactivated, the magnetic beads 533 are captured in blister 546 and the eluted nucleic acid solution is transferred to blister 548.

[0166] A first-stage PCR master mix from injection channel 515g is mixed with the nucleic acid sample in blister 548. Optionally, the mixture is mixed by forcing the mixture through channel 553 between 548 and 564. After several mixing cycles, the solution is contained within blister 564, where a pellet of first-stage PCR primers is provided, at least one primer set is provided for each target, and first-stage multiplex PCR is performed. If RNA targets are present, a reverse transcription (RT) step may be performed prior to or simultaneously with the first-stage multiplex PCR. Temperature cycling of first-stage multiplex PCR in the FilmArray® device is illustratively performed for between 15-20 cycles, although other levels of amplification may be desirable depending on the requirements of a particular application. The first-stage PCR master mix may be any of a variety of master mixes, as known in the art. In one example, the first-stage PCR master mix may be any of the chemicals disclosed in U.S. Pat. No. 9,932,634, incorporated herein by reference, for use in PCR protocols taking 20 seconds or less per cycle.

[0167] After the first-stage PCR has proceeded for a desired number of cycles, the sample can be diluted, illustratively by forcing most of the sample back into blister 548, leaving only a small amount in blister 564, and adding second-stage PCR master mix from injection channel 515i. Alternatively, dilution buffer from 515i can be transferred to blister 566 and then mixed with the amplified sample in blister 564 by moving the liquid back and forth between blisters 564 and 566. If desired, the dilution can be repeated several times using dilution buffer from injection channels 515j and 515k, or injection channel 515k can illustratively be primed for sequencing or other post-PCR analysis, and then second-stage PCR master mix can be added to some or all of the diluted amplified sample from injection channel 515h. It is understood that the level of dilution may be adjusted by varying the number of dilution steps, or by varying the percentage of sample that is discarded before mixing with dilution buffer, or components for amplification, illustratively a second-stage PCR master mix that includes, among other things, polymerase, dNTPs, and a suitable buffer (although in non-PCR amplification methods, other components may be suitable). If desired, this mixture of sample and second-stage PCR master mix may be preheated in blister 564 before transfer to second-stage well 582 for second-stage amplification. Such preheating may obviate the need for hot start components (antibodies, chemicals, or otherwise) in the second-stage PCR mixture.

[0168] In one embodiment, the exemplary second-stage PCR master mix is ​​incomplete and lacks a primer pair, and each of the 102 second-stage wells 582 is pre-filled with a specific PCR primer pair. In other embodiments, the master mix does not contain other components (e.g., polymerase, Mg 2+etc.), and the missing components may be pre-loaded in the array. Optionally, the second-stage PCR master mix may be missing other reaction components, and these components may be pre-loaded in the second-stage wells 582 as well. Each primer pair may be similar or identical to the first-stage PCR primer pair, or may be nested within the first-stage primer pair. Transfer of the sample from blister 564 to second-stage wells 582 completes the PCR reaction mixture. Once the high density array 581 is filled, the individual second-stage reactions are sealed in their respective second-stage blisters by any number of means, as known in the art. Exemplary methods for filling and sealing the high density array 581 without cross-contamination are discussed in U.S. Pat. No. 8,895,295, previously incorporated by reference. Illustratively, the various reactions within the wells 582 of the high density array 581 are thermally cycled simultaneously or individually, illustratively using one or more Peltier devices, although other means for thermal cycling are known in the art.

[0169] In certain embodiments, the second-stage PCR master mix includes the dsDNA binding dye LCGreen® Plus (BioFire Diagnostics, LLC) to generate a signal indicative of amplification. However, it is understood that this dye is merely exemplary and that other signals may be used, including other dsDNA binding dyes and probes that are fluorescently, radioactively, chemiluminescently, enzymatically, etc. labeled, as known in the art. Alternatively, wells 582 of array 581 may be provided in a signal-free condition and results reported through subsequent processing.

[0170] When air pressure is used to move material within the pouch 510, in one embodiment, a "bladder" may be utilized. Bladder assembly 810, a portion of which is shown in FIGS. 2-3, illustratively includes a bladder plate 824 housing a plurality of inflatable bladders 822, 844, 846, 848, 864, and 866, each of which may be individually inflatable by a compressed gas source. Because bladder assembly 810 receives compressed gas and may be used multiple times, bladder assembly 810 may be made from a stiffer or thicker material than the pouch. Alternatively, bladders 822, 844, 846, 848, 864, and 866 may be formed from a series of plates secured together with gaskets, seals, valves, and pistons. Other arrangements are within the scope of the invention. Alternatively, an array or mechanical actuators and seals may be used to seal the channels and direct the movement of liquid between blisters. Mechanical seal and actuator systems that may be adapted for use with the devices described herein are described in detail in U.S. Patent Application Publication No. 2019-0344269, previously incorporated by reference in its entirety.

[0171] The success of the secondary PCR reaction depends on the template generated by the multiplex first-stage reaction. Typically, PCR is performed with highly pure DNA. Methods such as phenol extraction or commercially available DNA extraction kits provide highly pure DNA. Samples processed through pouch 510 may need to be adjusted to compensate for less pure preparations. PCR may be inhibited by components of the biological sample, which are potential barriers. Illustratively, hot start PCR, higher concentrations of Taq polymerase enzyme, adjustment of MgCl2 concentration, adjustment of primer concentration, addition of modified enzymes resistant to inhibitors, and addition of adjuvants (such as DMSO, TMSO, or glycerol) can be optionally used to compensate for lower nucleic acid purity. It is understood that while purity issues are likely to be more of a concern in the first-stage amplification, similar adjustments can be provided in the second-stage amplification as well.

[0172] When pouch 510 is placed in device 800, bladder assembly 810 is pressed against one side of pouch 510 such that when a particular bladder is inflated, liquid is pressurized from a corresponding blister in pouch 510. Bladder assembly 810 may have bladders corresponding to many of the blisters of pouch 510, as well as additional pneumatic actuators, such as bladders or pneumatically actuated pistons, corresponding to various channels of pouch 510. Figures 2-3 show an exemplary plurality of pistons or hard seals 838, 843, 852, 853, and 865 corresponding to channels 538, 543, 553, and 565 of pouch 510, as well as seals 871, 872, 873, and 874 that minimize backflow into fitment 590. Hard seals 838, 843, 852, 853, and 865 form pinch valves to pinch off and close off corresponding channels when activated. To confine liquid within a particular blister of the pouch 510, a hard seal is activated over the channels leading to and from the blister, and the actuator acts as a pinch valve to pinch the channel closed. Illustratively, to mix two volumes of liquid in different blisters, a pinch valve actuator sealing the connecting channel is activated and a pneumatic bladder over the blister is alternately compressed, pressurizing the liquid back and forth through the channel connecting the blisters and mixing the liquid therein. The pinch valve actuator may be of various shapes and sizes and may be designed to pinch off two or more channels simultaneously. While pneumatic actuators are discussed herein, it is understood that other methods of applying pressure to the pouch are contemplated, including various electromechanical actuators such as linear stepper motors, motor-driven cams, rigid paddles driven by air pressure, hydrodynamic or electromagnetic forces, rollers, rocker arms, and in some cases, cocked springs. Additionally, there are various ways of reversibly or irreversibly closing a channel in addition to applying normal pressure to the axis of the channel.These include twisting the bag through the channel, heat sealing, rotating actuators, and sealing various physical valves to the channel such as butterfly valves and ball valves. Additionally, a small Peltier device or other temperature control device can be placed adjacent to the channel and set to a temperature sufficient to freeze the liquid, effectively forming a seal. It is also contemplated that while the design of FIG. 1 is adapted for an automated device characterized by an actuator element being placed on each of the blisters and channels, the actuators can remain stationary and the pouch 510 can be modified such that a small number of actuators can be used for several of the processing stations, including processing stations for sample disruption, nucleic acid capture, first and second stage PCR, and other applications of the pouch 510 such as immunoassays and immunoPCR. Rollers acting against the channels and blisters can prove particularly useful in designs where the pouch 510 is translated between stations. Thus, while pneumatic actuators are used in the embodiments of the present disclosure, it is understood that when the term "pneumatic actuator" is used herein, other actuators and other methods of providing pressure may be used depending on the design of the pouch and the device.

[0173] Returning to FIG. 2, each pneumatic actuator is connected to a compressed gas source 895 via a valve 899. While only a few hoses 878 are shown in FIG. 2, it will be understood that each pneumatic fitting is connected to a compressed gas source 895 via a hose 878. The compressed gas source 895 may be a compressor, or alternatively, the compressed gas source 895 may be a compressed gas cylinder, such as a carbon dioxide cylinder. Compressed gas cylinders are particularly useful when portability is desired. Other compressed gas sources are within the scope of the present invention. For example, similar pneumatic controls may be provided, or other actuators, servos, etc. may be provided, for control of liquid movement in the pouches described herein.

[0174] Also, several other components of the device are connected to a compressed gas source 895. A magnet 850 mounted on a second side 814 of the support member 802 is illustratively deployed and retracted using gas from the compressed gas source 895 via a hose 878, although other methods of moving the magnet 850 are known in the art. The magnet 850 is located in a recess 851 in the support member 802. It is understood that the recess 851 can be a passageway through the support member 802 such that the magnet 850 can contact the blister 546 of the pouch 510. However, depending on the material of the support member 802, it is understood that the recess 851 need not extend all the way through the support member 802, so long as the magnet 850 is close enough to provide a sufficient magnetic field at the blister 546 when the magnet 850 is deployed, and does not significantly affect any magnetic beads 533 present in the blister 546 when the magnet 850 is fully retracted. While reference is made to retracting the magnet 850, it is understood that an electromagnet may be used and that activation and deactivation of the electromagnet may be achieved by controlling the flow of electricity through the electromagnet. Thus, while removal or retraction of the magnet is discussed herein, it is understood that these terms are broad enough to encompass other methods of removing the magnetic field. It is understood that the pneumatic connections may be pneumatic hoses or pneumatic air manifolds, thus reducing the number of hoses or valves required. It is understood that similar magnets and methods for activating the magnets may be used in other embodiments.

[0175] Additionally, the various pneumatic pistons 868 of pneumatic piston array 869 are connected via hoses 878 to a compressed gas source 895. While only two hoses 878 are shown connecting the pneumatic pistons 868 to the compressed gas source 895, it will be understood that each of the pneumatic pistons 868 is connected to the compressed gas source 895. Twelve pneumatic pistons 868 are shown.

[0176] A pair of temperature control elements are mounted on the second side 814 of the support member 802. As used herein, the term "temperature control element" refers to a device that adds heat to or removes heat from a sample. Examples of temperature control elements include, but are not limited to, heaters, coolers, Peltier devices, resistive heaters, inductive heaters, electromagnetic heaters, thin film heaters, printed element heaters, positive temperature coefficient heaters, and combinations thereof. A temperature control element may include multiple heaters, coolers, Peltiers, and the like. In an embodiment, a given temperature control element may include more than one type of heater or cooler. For example, an example of a temperature control element may include a Peltier device with separate resistive heaters applied to the top and / or bottom surfaces of the Peltier. While the term "heater" is used throughout this specification, it is understood that other temperature control elements may be used to regulate the temperature of the sample.

[0177] As discussed above, first-stage heater 886 may be arranged to heat and cool the contents of blister 564 in first-stage PCR. As seen in Figure 2, second-stage heater 888 may be arranged to heat and cool the contents of second-stage blisters of array 581 of pouch 510 in second-stage PCR. However, it will be understood that these heaters may be used for other heating purposes as well, and other heaters may be included as appropriate for a particular application.

[0178] As discussed above, while Peltier devices that thermally cycle between two or more temperatures are useful for PCR, in some embodiments it may be desirable to maintain the heater at a constant temperature. Illustratively, this can be used to reduce run times by eliminating the time required to change heater temperature beyond the time required to change sample temperature. Such provisions can also improve the electrical efficiency of the system, since it is necessary to simply thermally cycle smaller samples and sample vessels rather than much larger (larger thermal mass) Peltier devices. For example, the device may include multiple heaters (i.e., two or more) positioned relative to the pouch to achieve thermal cycling, e.g., at set temperatures for annealing, extension, denaturation. Two heaters may be sufficient for many applications. In various embodiments, the heater is movable, the pouch is movable, or the liquid is movable relative to the heater to achieve thermal cycling. Illustratively, the heaters may be positioned in a linear, annular arrangement, or similar. Suitable heater types are discussed above with reference to first-stage PCR.

[0179] When fluorescence detection is desired, an optical array 890 may be provided. As shown in FIG. 2, the optical array 890 includes a light source 898, illustratively a filtered LED light source, filtered white light, or laser illumination, and a camera 896. The camera 896 illustratively has multiple photodetectors, each corresponding to a second-stage well 582 in the pouch 510. Alternatively, the camera 896 can acquire an image including all of the second-stage wells 582, and the image can be divided into separate regions corresponding to each of the second-stage wells 582. Depending on the design, the optical array 890 may be stationary, or the optical array 890 may be mounted on one or more motors and disposed on a mover that moves to obtain a signal from each individual second-stage well 582. It is understood that other arrangements are possible. Some embodiments of the second-stage heater provide a heater on the side of the pouch 510 opposite to that shown in FIG. 2. Such arrangements are merely exemplary and may be determined by spatial constraints within the device. Photodetectors and heaters may be provided on either side of array 581, provided that second-stage reaction zone 580 is provided with an optically transparent material.

[0180] As shown, computer 894 controls valve 899 of compressed gas source 895, and therefore all of the pneumatic systems of device 800. Additionally, in other embodiments, many of the pneumatic systems in the device may be replaced with mechanical actuators, pressure application means, and the like. Computer 894 also controls heaters 886 and 888, as well as optical array 890. Each of these components is illustratively electrically connected via cable 891, although other physical or wireless connections are within the scope of the present invention. It will be appreciated that computer 894 may be housed within device 800 or may be provided external to device 800. Additionally, computer 894 may include an integrated circuit board that controls some or all of the components, and may also include an external computer, such as a desktop or laptop PC, for receiving and displaying data from the optical array. An interface may be provided, illustratively a keyboard interface including keys for inputting information and variables such as temperature, cycle time, and the like. Also illustratively a display 892 is provided. The display 892 may be, for example, an LED, LCD, or other such display.

[0181] Other devices known in the art teach PCR in a sealed flexible container. See, for example, U.S. Patent Nos. 6,645,758, 6,780,617, and 9,586,208, which are incorporated herein by reference. However, for example, cell lysis in a sealed PCR container may improve ease of use and safety, especially when the sample to be tested may contain a biohazard. In the embodiment illustrated herein, waste from cell lysis, as well as waste from all other steps, remains in the sealed pouch. It is further understood that the contents of the pouch can be removed for further testing.

[0182] Returning to Figure 2, device 800 includes a support member 802, which may form a wall of the casing or may be mounted within the casing. Device 800 may also optionally include a second support member (not shown) that is movable relative to support member 802 to allow for insertion and removal of pouch 510. Illustratively, a lid may cover pouch 510 once pouch 510 has been inserted into device 800. In another embodiment, both support members may be fixed and pouch 510 may be secured in place by other mechanical means or by air pressure.

[0183] In the illustrative example, heaters 886 and 888 are mounted on support member 802. However, it is understood that this arrangement is merely illustrative and that other arrangements are possible. Exemplary heaters include Peltier and other block heaters, resistive heaters, electromagnetic heaters, and thin film heaters, as are known in the art, for thermally cycling the contents of blister 864 and second-stage reaction zone 580. Bladder plate 810, along with bladders 822, 844, 846, 848, 864, 866, hard seals 838, 843, 852, 853, and seals 871, 872, 873, 874, form bladder assembly 808, which illustratively may be mounted on a movable support structure that is movable toward pouch 510, where a pneumatic actuator is disposed in contact with pouch 510. When pouch 510 is inserted into device 800 and the flexible support member is moved towards support member 802, the various blisters of pouch 510 are positioned adjacent to the various bladders of bladder assembly 810 and the various seals of assembly 808 such that actuation of a pneumatic actuator can push liquid out of one or more of the blisters of pouch 510 or form a pinch valve with one or more channels of pouch 510. The relationship between the blisters and channels of pouch 510 and the bladders and seals of assembly 808 is illustrated in more detail in FIG.

[0184] Isolation, enrichment, characterization, and / or identification of microorganisms in a sample The present invention provides methods, systems, and devices for the isolation, enrichment, characterization, and / or identification of microorganisms in a sample. In one embodiment, the microorganism is a bacterium. In another embodiment, the microorganism is a fungal organism (e.g., a yeast or mold). In a further embodiment, the microorganism is a parasite. In another embodiment, the microorganism may be a combination of microorganisms selected from the group consisting of bacteria, yeast, mold, and parasites. The methods, systems, and devices may be particularly useful for the isolation, characterization, and / or identification of microorganisms from complex samples such as blood, urine, or cerebrospinal fluid. In a preferred aspect, the methods, systems, and devices of the present invention may be used to isolate, characterize, and / or identify microorganisms obtained directly from blood, for example, to rapidly determine whether a patient is septic or pre-septic.

[0185] As used herein, "obtained directly from blood" or "obtained directly from whole blood" in the context of determining the presence of a microorganism present in a blood sample means determining the presence of a microorganism by concentrating and / or isolating the microorganism from the whole blood and then identifying the microorganism. "Whole blood" is blood (e.g., human blood) without any of its components having been separated or removed as it would be found in the circulatory system. Blood with added anticoagulant is also commonly referred to as whole blood. Microorganisms may be enriched and / or isolated from whole blood, preferably without a preconcentration and / or preisolation blood culture step to increase the number of microorganisms in the sample. Microorganisms may be enriched and / or isolated from blood, preferably after a short (e.g., <5 hours, <4 hours, <3 hours, <2 hours, or <1 hour) culture step to increase the number of microorganisms in the sample. After enrichment and / or isolation of the microorganisms, the microorganisms may be suitably identified by several techniques, including, but not limited to, one or more of molecular (i.e., nucleic acid-based), phenotypic, proteomic, optical, or culture-based tests. After enrichment and / or isolation of the microorganisms, the microorganisms may be suitably cultured for a short period of time (<5 hours, <4 hours, <3 hours, <2 hours, or <1 hour (e.g., 3 hours)) to increase the number of enriched / isolated fractions. However, culture may suitably not be necessary in the methods and systems described herein. For example, the methods described herein may suitably work for all bacterial and fungal organisms of interest, including, but not limited to, fastidious organisms that typically do not grow well or rapidly in blood culture, aerobic and anaerobic organisms that may require different culture conditions, and organisms that may require different media formulations for growth and detection.

[0186] Characterization and / or identification of microorganisms in a concentrated sample of microorganisms (e.g., centrifugation pellet) may suitably not include precise species identification. Characterization encompasses broad categorization or classification of biological particles as well as actual identification of a single species. As used herein, "identification" refers to determining the family, genus, species, and / or lineage to which a microorganism belongs. For example, identification of microorganisms isolated from a biological sample (e.g., blood, urine, or cerebrospinal fluid) to the family, genus, species, and / or lineage level.

[0187] The methods, systems, and devices described herein allow for characterization and / or identification of microorganisms more quickly than the prior art, resulting in more rapid diagnosis (e.g., in subjects with or suspected of having sepsis). The steps involved in the methods of the present invention can be performed within a very short time frame from obtaining a sample for characterization / identification of the microorganism to obtaining clinically relevant and available information. In certain embodiments, the methods of the present invention can be performed within less than about 120 minutes, such as within about 110 minutes, about 100 minutes, about 95 minutes, about 90 minutes, about 85 minutes, about 80 minutes, about 75 minutes, about 70 minutes, about 65 minutes, about 60 minutes, about 55 minutes, about 50 minutes, about 45 minutes, about 40 minutes, about 35 minutes, about 30 minutes, about 25 minutes, about 20 minutes, about 15 minutes, about 10 minutes, about 5 minutes, about 4 minutes, about 3 minutes, about 2 minutes, about 1 minute, or within any range of, between, or including the above time points. In a preferred embodiment, the method of the present invention can be performed within less than about 90 minutes (e.g., about 75 minutes). For example, the elapsed time from the time a whole blood sample is collected from a patient suspected of having sepsis to the completion of analysis and positive identification of infectious agents (if present) can be less than about 90 minutes in many scenarios. When more rapid molecular analysis systems become available, sampling turnaround times can be significantly reduced. While sepsis and whole blood are used in the previous examples, similar times may be achievable in other complex sample types where low titer organisms are concentrated from large volumes of blood or other sample types. The extraordinary rapidity of the method of the present invention represents an improvement over previous methods. The method can be used to characterize and / or identify any microorganism as described herein.

[0188] Exemplary workflows associated with the methods, systems, and devices described herein are simple and minimize manipulation of samples, lysates, and microorganisms. For example, a sample can be mixed with a differential lysis buffer in a single tube for lysis and isolation of the microorganisms. In one embodiment, the microorganisms can be recovered from a single tube in a manner that segregates the microbial pellet from the lysate, thereby reducing the risk of handling potentially infectious materials and / or contaminating the sample. In addition, the methods of the present invention can be fully automated, thereby further reducing the risk of handling infectious materials and / or contaminating the sample.

[0189] FIG. 5 is a schematic diagram of one embodiment of components useful in the methods, systems, and devices described herein. The exemplary method includes a limited number of components, a limited number of steps, and can be completed within about 20-75 minutes from contact of the sample and the differential lysis buffer, providing a simplified workflow and faster time to results. The exemplary method of FIG. 5 includes obtaining a sample 5000 (e.g., a whole blood sample, a urine sample, a cerebrospinal fluid sample, or an environmental sample), preparing a lysate, recovering microbial cells from the lysate, and characterizing and / or identifying microorganisms in the sample. In one embodiment, the sample 5000, which may be a blood sample, may be provided in a standard blood collection tube (e.g., a vacutainer, etc.) with or without an anticoagulant. In one embodiment, the sample 5000 and the differential lysis buffer may be combined for lysis of substantially all (e.g., >90%) of the non-microbial cells in the sample 5000. In an exemplary embodiment, the lysate may be prepared in a specially designed centrifugal concentrator 5010. In one embodiment, the differential lysis buffer may be provided in the centrifugal concentrator 5010, and lysis of non-microbial cells in the sample may be initiated simply by adding the sample 5000 to the centrifugal concentrator 5010, thereby combining the sample and the differential lysis buffer in the centrifugal concentrator 5010. In another embodiment, the sample 5000 is mixed with the differential lysis buffer and then placed in the centrifugal concentrator 5010, e.g., pipetted into the centrifugal concentrator as a mixture. After combination, the differential lysis buffer and the sample are combined for a period of time (e.g., 1-5 minutes) to obtain a lysate. In one embodiment, the microorganisms may be recovered from the lysate by centrifugation, filtration, etc. In the case of centrifugation, the microbial cells may be pelleted in the centrifugal concentrator 5010 by centrifuging the centrifugal concentrator at about 1,000×g to about 20,000×g for a period of time ranging from about 4-10 minutes.In an exemplary embodiment, the recovered microbial cells may be added from the centrifugal concentrator 5010 to an analyzer 5020 designed to characterize and / or identify the microorganisms in the sample at a clinically relevant level. Characterization and / or identification of the microorganisms in the illustrated analyzer 5020 can be performed quickly (e.g., in about 15-60 minutes). However, the illustrated analyzer is merely exemplary. For example, in some embodiments, the microorganisms can be characterized and / or identified by sequencing (e.g., next generation sequencing).

[0190] sample Samples that may be tested by the methods and systems described herein may include both clinical and non-clinical samples in which the presence and / or growth of a microorganism is or may be suspected, as well as samples of materials that are routinely or occasionally tested for the presence of a microorganism. The amount of sample utilized may vary greatly due to the versatility and / or sensitivity of the method. One advantage of the methods and systems described herein is that complex sample types, such as, for example, blood, body fluids, and / or other opaque substances, may be directly tested using the system with little or no extensive pretreatment.

[0191] "Sample" means an animal; a tissue or organ from an animal, including, but not limited to, a human animal; a cell (either a cell within a subject taken directly from the subject (e.g., a human or non-human animal) or a cell maintained in culture or from a cultured cell line); a cell lysate (or a lysate fraction) or cell extract; a solution containing one or more molecules (e.g., polypeptides or nucleic acids) derived from a cell, cellular material, or viral material; or a solution containing a non-naturally occurring nucleic acid that is assayed as described herein. Samples that may be tested by the methods and systems described herein may include both clinical and non-clinical samples in which the presence and / or growth of a microorganism is or may be suspected, as well as samples of materials that are routinely or occasionally tested for the presence of a microorganism. Clinical samples that may be tested include any type of sample that is typically tested in a clinical or research laboratory, including, but not limited to, blood, serum, plasma, blood fractions, joint fluid, urine, semen, saliva, stool, cerebrospinal fluid, stomach contents, vaginal secretions, tissue homogenates, bone marrow aspirates, bone homogenates, sputum, aspirates, swabs and swab rinsates, other bodily fluids, blood products (e.g., platelets, serum, plasma, white blood cell fractions, etc.), donor organ or tissue samples, etc. Some specimen samples that may be cultured and then tested may include blood, serum, plasma, platelets, red blood cells, white blood cells, blood fractions, joint fluid, urine, nasal samples, semen, saliva, stool, cerebrospinal fluid, stomach contents, vaginal secretions, tissue homogenates, bone marrow aspirates, bone homogenates, sputum, aspirates, swabs and swab rinsates, other bodily fluids, etc. For example, in some embodiments, it may be an option to subject a sample, such as blood, from a subject to a limited incubation step (e.g., in the range of 1 minute to 4 hours) prior to testing to increase the level of detectable microorganisms in the sample. In another option, a sample, such as blood, may be cultured prior to selective lysis and recovery of microorganisms, the microorganisms may be cultured during lysis and recovery, or the microorganisms may be cultured from a pellet recovered (e.g., by centrifugation) from a sample that has been selectively lysed (e.g., by growing the organisms in liquid medium or on a solid plate).Culturing of microorganisms (especially bacteria and fungi) can preferably be more rapid when the cell concentration is higher. Culturing from harvested or concentrated microorganisms (e.g., from the pellet obtained from a centrifugation step) can preferably be more rapid than blood cultures. Also, culturing from harvested or concentrated microorganisms can preferably remove antibiotics and defensins that may be present in the blood and promote more rapid growth.

[0192] The present invention finds use in research, as well as veterinary and medical applications. Suitable subjects from which clinical samples can be obtained are generally mammalian subjects, but can be any animal. The term "mammal" as used herein includes, but is not limited to, humans, non-human primates, cows, sheep, goats, pigs, horses, cats, dogs, rabbits, rodents (e.g., rats or mice), and the like. Human subjects include neonatal, infant, juvenile, adult, and geriatric subjects. Subjects from which samples can be obtained include, but are not limited to, mammals, birds, reptiles, amphibians, and fish.

[0193] Non-clinical samples that can be tested also include substances including, but not limited to, food, beverages, pharmaceuticals, cosmetics, water (e.g., potable, non-potable, and wastewater), seawater ballast, air, soil, wastewater, plant material (e.g., seeds, leaves, stems, roots, flowers, fruits), biological weapon samples, and the like. Samples may also include environmental samples such as, but not limited to, soil, air monitoring system samples (e.g., material trapped in air filter media), surface swabs, and vectors (e.g., mosquitoes, ticks, fleas, and the like). The method is also well suited for real-time testing, particularly for monitoring contamination levels, process control, quality control, and the like in industrial settings. In a preferred embodiment of the invention, the sample is obtained from a subject (e.g., a patient) having or suspected of having a microbial infection. In one embodiment, the subject has or is suspected of having sepsis, e.g., bacteremia or fungemia. Preferably, the sample may be a blood sample that is collected from the subject and then directly tested. That is, the sample is a whole blood sample that has not been spiked into blood medium and has not been processed or cultured or diluted prior to testing. In another embodiment, the sample may be obtained from a blood culture grown from a sample of the patient's blood, e.g., a BacT / ALERT® blood culture. The blood culture sample may be obtained from a positive blood culture, e.g., a blood culture that shows the presence of a microorganism. In certain embodiments, the sample may be taken from a positive blood culture within a short time after it turns positive, e.g., within about 6 hours, e.g., within about 5 hours, about 4 hours, about 3 hours, or about 2 hours, or within about 60 minutes, e.g., within about 55 minutes, about 50 minutes, about 45 minutes, about 40 minutes, about 35 minutes, about 30 minutes, about 25 minutes, about 20 minutes, about 15 minutes, about 10 minutes, about 5 minutes, about 4 minutes, about 3 minutes, about 2 minutes, or about 1 minute. In one embodiment, the sample may be taken from a culture in which the microorganism is in logarithmic phase growth. In another embodiment, the sample may be taken from a culture in which the microorganism is in stationary phase. In some embodiments, a whole blood sample may be provided as part of the method within one hour of the whole blood sample being taken from the patient.In yet another embodiment, the sample may be or include blood that has been cultured for a period of time that is typically shorter than the time required to obtain a positive blood culture result (e.g., in the range of 1 minute to 4 hours). In some embodiments, the sample is provided at room temperature for use in the method, while in other embodiments, the sample is cooled after it is obtained from the patient and before it is provided for use in the method. For example, the sample may be refrigerated after it is obtained from the patient until the method can be performed.

[0194] The present invention provides high sensitivity for the detection and identification of microorganisms. Illustratively, this allows for the detection and identification of microorganisms without first having to go through a step of liquid culture, followed by isolating the microorganisms and growing them on a solid or semi-solid medium, and sampling the growing colonies. Thus, in one embodiment of the present invention, the sample is not obtained from a liquid culture, or from a microbial (e.g., bacteria, yeast, or mold) colony grown on a solid or semi-solid surface. To facilitate the identification of likely BSIs, in some embodiments, the method includes a step of lysing the sample without culturing it after it is obtained from the patient. In some embodiments, the methods described herein can be used even with patients who have been treated with antimicrobial agents before the collection of the blood sample. Patients who are hospitalized with symptoms consistent with sepsis often have antimicrobial agents started immediately, after which sepsis can be definitively included or excluded. While such treatment protocols are consistent with the standard of care, antimicrobial agents may interfere with blood cultures used in classical sepsis diagnosis. Surprisingly, the methods described herein can further be used to diagnose sepsis in patients on antibiotic treatment when intact microbial cells are still present in the blood.

[0195] The volume of the sample should be large enough to generate a pellet of microorganisms that can be analyzed after the separation step of the method of the present invention is performed. The appropriate volume will depend on the source of the sample, the expected level of microorganisms in the sample, and the analytical method utilized to characterize and identify the microorganisms. For example, whole blood from a patient with a BSI typically has a microbial load of about 1-100 cfu / ml (e.g., <1-10 cfu / ml). In general, the sample size can be about 50 ml, about 40 ml, about 30 ml, about 20 ml, about 15 ml, about 10 ml, about 5 ml, about 4 ml, about 3 ml, or about 2 ml (e.g., about 10 ml). In certain embodiments, the sample size can be about 1 ml, e.g., about 0.75 ml, about 0.5 ml, or about 0.25 ml. In certain embodiments, such as where the separation is performed on a microscale, the sample size may be less than about 200 μl, e.g., less than about 150 μl, about 100 μl, about 50 μl, about 25 μl, about 20 μl, about 15 μl, about 10 μl, or about 5 μl. In some embodiments (e.g., when the sample is expected to contain a small number of microorganisms), the sample size may be about 100 ml or more, e.g., about 250 ml, about 500 ml, about 750 ml, or about 1000 ml or more. Positive blood cultures will contain higher levels of microorganisms per ml, and therefore, smaller volumes of blood medium may be used compared to whole blood.

[0196] While much of the discussion herein relates specifically to whole blood, the methods, systems, and devices described herein may be used with other sample types, as described in the definition of "sample" above. Two specific examples of additional sample types are urine and cerebrospinal fluid (CSF). Urine and CSF often contain white blood cells (WBCs) during infection, which may harbor intracellular pathogens. These WBCs may be produced when fighting an infection, or in the case of CSF, many pathogens may gain access to the brain / spinal column by hiding inside the WBCs or other blood cells, and then cross the blood-brain barrier. Blood cells harboring pathogens (but not pathogen cells) can be selectively lysed with the differential lysis buffers disclosed herein to release the intracellular pathogens and concentrate them into a pellet that may be substantially free of contaminating eukaryotic host DNA (e.g., contaminating host DNA may be reduced by >95%). In addition, epithelial cells of the bladder can slough off during infection, shedding pathogen-carrying cells and preventing the spread of infection as a preventative measure. Similar to white blood cells, these epithelial cells can be lysed by the differential lysis buffer disclosed herein, and intact pathogen cells can be concentrated in the pellet without contamination from bladder cells.

[0197] As discussed in more detail elsewhere herein, the recovered pathogen cells may be lysed and nucleic acid from the pathogen cells may be recovered for analysis. Because the pathogen cells are isolated without significant host cell DNA contamination, the recovered pathogen nucleic acid is suitable for downstream molecular assays to characterize and / or identify the pathogen. In some embodiments, the pathogen cells may be used for downstream characterization and / or identification of the pathogen by molecular methods (e.g., by PCR amplification of the pathogen DNA or RNA and identification of the amplified product), by genetic sequencing (e.g., by next generation sequencing techniques), or by mass spectrometry. The devices and methods described herein may remove many or all of the host cell components so that the pathogen signal can be identified by any of these methods.

[0198] Lysis step After providing or obtaining a sample, the next step in the exemplary method of the invention is to lyse non-microbial cells that may be present in the sample, such as blood cells and / or tissue cells or other eukaryotic host cells. In some embodiments, the method includes selectively lysing the cells to allow separation of the microorganisms from other components of the sample. Separation of the microorganisms from other components reduces interference during subsequent investigation steps. If non-microbial cells are not expected to be present in the sample or to interfere with the investigation steps, the lysis step may be omitted. In one embodiment, the cells to be lysed are the non-microbial cells present in the sample, and little or no microbial cells that may be present in the sample are lysed. However, in some embodiments, selective lysis of specific classes of microorganisms may be desirable and therefore can be performed according to the methods described herein and as known in the art. For example, classes of undesirable microorganisms can be selectively lysed, such as yeast being lysed while bacteria are not, or vice versa. In another embodiment, the desired microorganisms are lysed to isolate specific intracellular components of the microorganisms, such as cell membranes or organelles. In one embodiment, all of the non-microbial cells are lysed. In other embodiments, a portion of the non-microbial cells are lysed, e.g., enough cells to prevent interference with the interrogation step. Lysis of cells may be performed by any method known in the art, including but not limited to, addition of differential lysis buffers, sonication, and / or osmotic shock, effective to selectively lyse cells in the presence or absence of lysed microorganisms.

[0199] A differential lysis buffer is one that can selectively lyse one class of cells, e.g., non-microbial cells and / or some microbial cells (e.g., by solubilizing eukaryotic cell membranes), and cannot lyse another class of cells, e.g., a microorganism or type of microorganism. In one embodiment, the differential lysis buffer can include an aqueous medium, one or more detergents, a buffer substance, one or more salts, and can further include additional agents. In one embodiment, the differential lysis buffer can further include one or more enzymes (e.g., proteases). In one embodiment, the detergent may be a non-denaturing soluble detergent such as Triton® X-100 Triton® X-100-R, Triton® X-114, NP-40, Igepal® CA630, Arlasolve™ 200, BrijO10 (also known as Oleth-10, Brij96V, Brij97, Volpo 10NF, Volpo N10) (the Brij name is a registered trademark of Croda International Plc), CHAPS, octyl β-D-glucopyranoside, saponin, and nonaethylene glycol monododecyl ether (also known as C12E9, polydosenol, Brij35). In one embodiment, the detergent is a non-ionic surfactant. Examples of suitable non-ionic surfactants include, but are not limited to, Triton X-114, NP-40, Arlasolve 200, BrijO10, octyl β-D-glucopyranoside, saponin, nonaethylene glycol monododecyl ether, and combinations thereof. In a preferred embodiment, the non-ionic surfactant is a polyoxyethylene ether (POE ether). POE ethers are a class of non-ionic surfactants that may be used for cell membrane disruption. POE ethers consist of an alkyl chain, a hydrophilic portion consisting of "n" oxyethylene units, and a terminal -OH group. Suitable examples of POE ethers include, but are not limited to, Arlasolve 200 (poly(oxy-1,2-ethanediyl)), BrijO10 (and other Brij detergents), and nonaethylene glycol monododecyl ether (Brij 35).Optionally, denaturing solubilizing detergents such as sodium dodecyl sulfate, N-lauryl sarcosine, sodium deoxycholate, bile salts, hexadecyltrimethylammonium bromide, SB3-10, SB3-12, amidosulfobetaine-14, and C7BzO may be included. Optionally, solubilizing agents such as Brij98, Brij58, Brij35, Tween®80, Tween®20, Pluronic®L64, Pluronic®P84, non-detergent sulfobetaine (NDSB201), amphipol (PMAL-C8), and methyl-β-cyclodextrin may also be included. Typically, non-denaturing detergents and solubilizing agents are used at concentrations higher than their critical micelle concentration (CMC), while denaturing detergents may be added at concentrations lower than their CMC. For example, non-denaturing lytic detergents can be used at a concentration (final concentration after sample dilution) of about 0.010% to about 10%, for example, about 0.015% to about 1.0%, for example, about 0.05% to about 0.5%, for example, about 0.10% to about 0.30%. Enzymes that can be used in the differential lysis buffer include, but are not limited to, enzymes that digest nucleic acids and other membrane fouling materials (e.g., proteinase XXIII, DNase, neuraminidase, polysaccharidase, Glucanex®, and Pectinex®). In a specific embodiment, the differential lysis buffer does not contain DNase or is not used in combination with DNase. Other additives that can be used include, but are not limited to, reducing agents such as 2-mercaptoethanol (2-Me) or dithiothreitol (DTT), and stabilizing agents such as magnesium, pyruvate, and humectants.

[0200] The differential lysis buffer can be buffered at any pH suitable for lysing the desired cells, and will depend on several factors including, but not limited to, the type of sample, the cells to be lysed, and the detergent used. In some embodiments, the pH can be in the range of about 2 to about 13, e.g., about 6 to about 10, e.g., about 7 to about 9, e.g., about 7 to about 8. Suitable pH buffers may include any buffer capable of maintaining the pH within the desired range. In some embodiments, buffers may be used outside of their pH buffering range. Suitable examples of buffer substances may include, but are not limited to, about 0.005M to about 1.0M CAPS, CAPSO, CHES, CABS, and combinations thereof. In a specific example, the differential lysis buffer has the composition shown in Table 1 below.

[0201] [Table 1]

[0202] In the specific example illustrated in Table 1, the sample is about 10 ml of whole blood combined with about 30 ml of Differential Lysis Buffer.

[0203] CAPS is a buffering substance; CAPS has a pKa of about 10.4 at 25° C. and a typical buffering range of about 9.7-11.1. Before the differential lysis buffer is combined with the blood sample, the CAPS buffer is within its buffering range. However, after the differential lysis buffer is combined with the blood sample, the CAPS buffer in this example is well outside its buffering range (e.g., at a pH of about 7-8). Surprisingly, it has been found that using CAPS (and chemically similar buffers - e.g., CAPSO, CHES, and CABS) in the differential lysis buffer, which is outside its buffering range, can have a synergistic effect to improve lysis. Without being bound to one theory, it is believed that CAPS may be acting like a second detergent to help permeabilize and lyse non-microbial cells. For example, at a pH of about 7-8 (e.g., a pH of 7.6-8), the CAPS buffer becomes almost completely protonated and positively charged. According to the Henderson-Hasselbach equation, for example, in the pH range of about 7.0-8.0, the ratio of protonated to unprotonated CAPS species is about 250:1 or more. For CAPS buffer at pH of about 7.0-8.0, the ratio of protonated to unprotonated of about 250:1 or more is an example of what it means to be "substantially positively charged". Since cell membranes generally have a net negative charge, it is theorized that the positive charge of CAPS buffer may attract CAPS molecules to the surface of cells. CAPS has a phenyl ring that may insert into the hydrophobic membrane of non-microbial cells to aid in cell permeabilization. CAPSO, CHES, and CABS have similar structures to CAPS, and it is expected that CAPSO, CHES, and CABS, as well as combinations of CAPS, CAPSO, CHES, and CABS, and similar buffers, may provide similar results. CAPSO has a pKa of about 9.6 at 25° C. and a typical buffering range of about 8.9-10.3, CHES has a pKa of about 9.3 at 25° C. and a typical buffering range of about 8.6-10, and CABS has a pKa of about 10.7 at 25° C. and a typical buffering range of about 10-11.4.For CAPSO, CHES, and CABS, the Henderson-Hasselbach equation provides that in the example pH range of about 7.0-8.0, the ratio of protonated to unprotonated buffer species is within the ranges of about 500:1 or more (i.e., CABS at a pH of about 7.0-8.0), about 40:1 or more (i.e., CAPSO at a pH of about 7.0-8.0), and 20:1 or more (i.e., CHES at a pH of about 7.0-8.0). Thus, for CAPSO, CHES, and CABS at a pH of about 7.0-8.0, a ratio of protonated CAPSO to unprotonated CAPSO of about 40:1 or more, a ratio of protonated CHES to unprotonated CHES of about 20:1 or more, and a ratio of protonated CABS to unprotonated CABS of about 500:1 or more are each further examples of what is meant by "substantially positively charged". Also, one of skill in the art will appreciate that the buffer material used in the differential lysis buffer may suitably include a combination of CAPS, CAPSO, CHES, and CABS. For such combinations, any ratio of protonated species to unprotonated species of about 20:1 or more is a further example of what is meant by "substantially positively charged".

[0204] In one embodiment, the sample and differential lysis buffer are combined for a sufficient time for lysis to occur, e.g., about 1 second, about 2 seconds, about 3 seconds, about 4 seconds, about 5 seconds, about 10 seconds, about 15 seconds, about 20 seconds, about 25 seconds, about 30 seconds, about 40 seconds, about 50 seconds, or about 60 seconds, or about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 15 minutes, or about 20 minutes or more, e.g., about 1 second to about 20 minutes, about 1 second to about 5 minutes, or about 1 second to about 2 minutes. In one embodiment, up to 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% of the non-microbial cells in the sample can be lysed within 2 to 5 minutes of combining the sample with the differential lysis buffer. In some embodiments, the sample and differential lysis buffer are combined for a sufficient time for solubilization of the cell membrane to occur. Solubilization of the cell membrane of blood cells (i.e., non-microbial cells) is illustrated in FIG. 21. To demonstrate the effectiveness of blood cell lysis at room temperature, the OD500 absorbance (wavelength of 500 nm) of whole blood combined with several differential lysis buffer formulations was measured at various time points. The decrease in absorbance as a function of time illustrates the progress of lysis. A buffer / blood combination containing 0.125% BrijO10 and 50 mM CAPS (○) (i.e., the detergent and buffer concentrations after combining 10 ml of whole blood with 30 ml of differential lysis buffer) was ineffective for lysis, likely due to insufficient detergent concentration. Each of the other three buffers tested (0.15% BrijO10 and 100 mM CAPS (△), 0.25% BrijO10 and 10 mM CAPS (□), and 0.25% BrijO10 and 50 mM CAPS (◇)) was effective in lysing blood cells. The decrease in absorbance in these buffer / blood combinations illustrates that lysis was complete within 2 minutes in the 100 mM CAPS buffer and the 50 mM CAPS buffer, and within 3 minutes in the 10 mM CAPS buffer. The buffers containing 0.25% BrijO10 and 10 mM CAPS are the buffers exemplified in Table 1 above. As illustrated in FIG. 21, the lysis time will depend on the strength of the differential lysis buffer, e.g., the concentration of the detergent and / or the pH of the solution.In general, it is expected that weaker lysis buffers will require more time and greater dilution of the sample to completely or partially solubilize non-microbial cells. The strength of the differential lysis buffer can be selected based on the microorganisms known or suspected to be present in the sample. For microorganisms that are more susceptible to lysis, weaker differential lysis buffers can be used. Lysis can occur at temperatures of about 2°C to about 45°C, e.g., about 15°C to about 40°C, e.g., about 20°C to about 40°C.

[0205] In one embodiment, the differential lysis buffer can be loaded into a syringe and then the sample can be aspirated into the syringe so that the combination occurs in the syringe. In one embodiment, the sample and the differential lysis buffer can be provided in separate tubes and they can be combined by pouring one into the other. In one embodiment, the differential lysis buffer can be provided in a centrifugal concentrator and the sample can be aspirated into the centrifugal concentrator so that the combination and microbial recovery occurs in the centrifugal concentrator. In some embodiments, the mixing is performed by combining the sample and the differential lysis buffer in solution. In further embodiments, the mixing includes agitating the combined sample and the differential lysis buffer. For example, the sample and the differential lysis buffer can be combined in a centrifugal concentrator and mixed by tilting or gently shaking the centrifugal concentrator. In another example, a bead beater or sonicator can be used to agitate the combined sample and the differential lysis buffer.

[0206] In some embodiments, the lysis conditions (e.g., combination and / or combination time) and the separation and / or investigation steps may be sufficient to kill some or all of the microorganisms in the sample. The methods of the invention are highly versatile and do not require that the microorganisms be viable when isolation and identification are performed. In certain embodiments, some or all of the microorganisms may be dead, and death may occur before, during, and / or after the steps of the method are performed. In other embodiments, some or all of the microorganisms may be viable as a result of the separation step, allowing further cultivation of the microorganisms at incubation temperatures (e.g., about 37° C. for bacteria and about 32° C. for many fungal species) in an appropriate medium (e.g., bacterial or fungal medium). For example, the microorganisms may be viable after the separation step and then included in a separation technique to determine whether the microorganism is sensitive or resistant to one or more antibiotics, and preferably the growth of the microorganisms may not be affected by the use of a differential lysis buffer.

[0207] Separation Step After the sample is lysed, a separation step can be performed to separate the microorganisms from other components of the sample and concentrate the microorganisms into a pellet that can be examined for identification and characterization purposes. The separation does not need to be complete, i.e., 100% separation is not required. Illustratively, the separation of the microorganisms from other components of the sample is sufficient to allow the examination of the microorganisms without substantial interference from the other components. For example, the separation can result in a microbial pellet that is at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% or more pure. One contaminant that potentially confounds microbial identification directly from whole blood is human genomic DNA. In one exemplary embodiment, the inventors in this case have found that treatment of whole blood with the differential lysis buffer described herein is capable of removing 98% or more of human genomic DNA when microorganisms are subsequently pelleted from the blood lysate even without DNase treatment.

[0208] In one embodiment, separation is performed by a centrifugation step in which the sample (e.g., lysed sample) is placed in a centrifugal concentrator and the centrifugal concentrator container is centrifuged under conditions in which the microbial pellet on the bottom and / or sides of the container and other components of the sample (e.g., lysed cellular components) in the sample medium remain in the supernatant. This separation isolates the microorganisms from materials in the sample such as medium, cellular debris, human genomic DNA, and / or other components that may interfere with the investigation of the microorganism (e.g., by amplification and detection of nucleic acids specific for the microorganism). This separation isolates the microorganisms from the bulk volume of the sample, reducing the volume of the microbial portion and concentrating the microorganisms in a small volume (e.g., about 200 μl). In one embodiment, a differential lysis buffer is provided in the centrifugal concentrator and lysis is initiated by combining the sample and the differential lysis buffer for a time sufficient for lysis, followed by recovery of the microorganisms by centrifugation. In one embodiment, the centrifugal concentrator does not include a high density cushion, physical separator, or similar medium known in the art. Surprisingly, it has been found that a high density cushion is not necessary to provide sufficient separation and isolation of microorganisms from contaminating debris when used in conjunction with molecular techniques for identification or characterization.

[0209] In one embodiment of the present invention, the centrifugal concentrator is centrifuged in a swinging bucket rotor such that the microorganisms form a pellet directly at the bottom of the tube. The container is centrifuged at a sufficient acceleration for a sufficient time to pellet the microorganisms and / or separate them from other components of the sample. The acceleration of the centrifugation can be illustratively about 1,000×g to about 20,000×g, such as about 2,500×g to about 15,000×g, such as about 7,500×g to about 12,500×g, etc. The centrifugation time can be illustratively about 30 seconds to about 30 minutes, such as about 1 minute to about 15 minutes, such as about 1 minute to about 10 minutes. The centrifugation can be carried out at a temperature illustratively about 2° C. to about 45° C., such as about 15° C. to about 40° C., such as about 20° C. to about 30° C. In one embodiment, the centrifugal concentrator includes a closure, and the closure is applied to the container and a seal is formed, followed by centrifugation. The presence of the closure reduces the risks arising from handling microorganisms that are or may be infectious and / or harmful, as well as the risk of contaminating the sample. One advantage of the method of the present invention is the ability to carry out any one or more of the steps of the method (e.g., lysis, separation, investigation, and / or identification) with microorganisms in a sealed container (e.g., a hermetically sealed container). The method may include the use of an automated system, which avoids the health and safety risks associated with handling highly toxic microorganisms, for example, that arise with the recovery of microorganisms from samples in direct testing.

[0210] The centrifugal concentrator may be any container having a sufficient volume to hold the differential lysis buffer and the sample. In one embodiment, the container fits or can be fitted to a centrifuge rotor. Exemplarily, the volume of the container can be about 0.1 ml to about 100 ml, e.g., about 50 ml. If the separation is done on a microscale, the volume of the container can be about 2 μl to about 100 μl, e.g., about 5 μl to about 50 μl. In one embodiment, the container has a wider internal diameter at the upper portion for holding the sample and a narrower internal diameter at the lower portion where the microbial pellet is collected. A tapered internal diameter portion can connect the upper portion and the lower portion. Exemplarily, the tapered portion can have an angle of about 20° to about 70°, e.g., about 30° to about 60°. In one embodiment, the lower narrow portion is less than half the total height of the container, e.g., less than about 40%, about 30%, about 20%, or about 10% of the total height of the container. The container may have a closure device attached or may be fitted to receive a closure device (e.g., a cap) and the container may be sealed prior to centrifugation. In certain embodiments, the container is designed so that the microbial pellet can be easily retrieved from the container after separation, either manually or in an automated manner (so that the technician is not exposed to the container contents). For example, the container may include a removable or peelable portion that contains the pellet and can be separated from the rest of the container. In another embodiment, the container includes one or more structures that allow access to the pellet after separation, such as one or more ports or permeable surfaces for insertion of a syringe or other sampling device or for removing the pellet. In one embodiment, the container is a stand-alone container, i.e., a device for separating a single sample. In other embodiments, the container is part of a device that includes two or more centrifugal concentrators so that multiple samples can be separated simultaneously. In one embodiment, the apparatus includes 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 36, 42, 48, 60, 72, 84, 96, or more centrifugal concentrators.

[0211] In another embodiment, separation can be performed by a filtration step where the sample (e.g., a lysed sample) is placed in an apparatus fitted with a selective filter or filter set having a pore size that retains the microorganisms. Other examples of filtration include, but are not limited to, tangential flow filtration and / or buffer exchange, where the microorganisms are separated from the sample, the sample volume is reduced, and the microorganisms are concentrated. Suitable examples of filtration techniques that may be used in the methods described herein are illustrated in Figures 15-20. The retained microorganisms may be washed by gently passing a suitable buffer through the filter. The washed microorganisms may then be interrogated directly on the filter and / or recovered for interrogation by directly sampling the surface of the filter or by back-flushing the filter with a suitable aqueous buffer.

[0212] In one embodiment, the container may be a tube, e.g., a centrifuge tube. In another embodiment, the container may be a chip or a card. In one embodiment, the inventors have developed a centrifugal concentrator, and associated devices, systems, and methods, that may enable lysis of non-microbial cells and recovery of microbial cells to be performed in a single tube. Furthermore, a microbial pellet may be generated from the centrifugal concentrator, such that the supernatant is isolated and contained within the upper portion of the centrifugal concentrator. Specifically, the centrifugal concentrator, and associated devices, systems, and methods described herein allow users to separate microorganisms from a sample with fewer operations and only a single centrifugation step. Additionally, the centrifugal concentrator, and associated devices, systems, and methods described herein allow users to separate and test samples without handling microorganisms, thus avoiding the health and safety risks associated with handling highly toxic microorganisms.

[0213] 6A-6F, an embodiment of a centrifugal concentrator 5010 and elements of a centrifugal concentrator are illustrated. In one embodiment, the centrifugal concentrator is a centrifuge tube designed for the concentration of microorganisms from a sample by centrifugation. The centrifugal concentrator 5010 includes a tubular body 6002 and a closure cap 6006 at the proximal end 6001 of the tubular body 6002. In one embodiment, a protective cap 6004 designed to protect the tube during centrifugation may be placed at the distal end 6005 of the tubular body 6002. In one embodiment, a differential lysis buffer and a sample (e.g., a whole blood sample) may be added to the tubular body 6002 after removing the cap 6006; the contents may be sealed therein by replacing the cap 6006. Illustratively, the distal 6005 and proximal 6001 ends of the centrifugal concentrator 5010 are sealed during operation to prevent the release of putative biohazardous materials, however, as described in more detail below, the distal end 6005 of the tubular body 6002 may be selectively openable to allow the discharge of pelleted microorganisms from the concentrator.

[0214] In an exemplary embodiment, the centrifugal concentrator 5010 includes a plunger 6008. The plunger 6008 may be designed to perform several functions, including, but not limited to, collecting concentrated (e.g., pelleted) microorganisms at or near the distal end of the centrifugal concentrator 5010, opening the distal end 6005 of the centrifugal concentrator 5010, and ejecting the pelleted microorganisms from the distal end 6005 of the centrifugal concentrator 5010. The opening of the concentrator distal end 6005 and ejection of the microorganism pellet will be discussed in more detail below in connection with Figures 6D and 6E. In one embodiment, the plunger 6008 has a proximal end 6024 that includes an enlarged portion 6009 designed for manual manipulation of the plunger 6008. For example, the enlarged portion 6009 may be manipulated by the thumb, finger, or another portion of a user's hand, or a mechanical device, to actuate the plunger 6008 and eject the pellet. For example, the plunger 6008 may be depressed by the user's thumb to actuate the plunger and expel the pellets. In another embodiment, the plunger 6008 is actuated in a different manner, for example, by rotating along a threaded screw portion to lower the plunger 6008 and penetrate the centrifugal concentrator. In an exemplary embodiment, the plunger 6008 includes a pair of retaining members 6026 designed to hold the plunger in a fixed "up" position in a first direction and a "plunge" position in a second direction. In one embodiment, the retaining members 6026 interact with a corresponding pair of detents 6030 on the cap 6006 to hold the plunger in a fixed "up" position. In an exemplary embodiment, to plunge, the enlarged portion 6009 is grasped and used to twist the plunger relative to the cap to align the retaining members 6026 with the passageway 6032. In one embodiment, the passageway 6032 is designed to allow the plunger 6008 to be depressed and the microbial pellet to be expelled from the distal end 6005 of the centrifugal concentrator.

[0215] In one embodiment, the centrifugal concentrator 5010 may have essentially any volume sufficient to hold the differential lysis buffer and the sample. In one embodiment, the centrifugal concentrator 5010 fits or can be fitted into a centrifuge rotor. Illustratively, the volume of the centrifugal concentrator 5010 can be from about 0.1 ml to about 100 ml. In a specific embodiment, the centrifugal concentrator 5010 has a shape and internal volume similar to a standard 50 ml conical centrifuge tube and is compatible with centrifuge rotors (fixed angle and swinging bucket) designed to fit 50 ml conical centrifuge tubes. In one embodiment, the differential lysis buffer 6003 is provided within the centrifugal concentrator 5010. For example, optionally, about 20 to 40 ml (e.g., about 30 ml) of a differential lysis buffer described herein may be provided within the centrifugal concentrator 5010. While the centrifugal concentrator 5010 may include a differential lysis buffer 6003, illustratively the centrifugal concentrator may not be provided with a high density cushion, regardless of whether a differential lysis buffer is provided in the centrifugal concentrator 5010. In one embodiment, the cap 6006 of the centrifugal concentrator 5010 may optionally include a septum 6007 (e.g., a rubber septum) or similar structure that may allow a sample (e.g., a whole blood sample) to be added to the centrifugal concentrator 5010 without having to remove the closure cap 6006. This may be particularly useful given that many of the samples intended to be used with the differential lysis buffer and centrifugal concentrators may be biohazardous and / or infectious. In some embodiments, the sample is mixed with the differential lysis buffer and aseptically filled into the centrifugal concentrator through the septum 6007. This reduces potential contamination of the sample prior to analysis.

[0216] FIG. 6B shows another view of the centrifugal concentrator 5010. In view of FIG. 6B, the external protective cap 6004 has been removed from the distal end of the tubular body 6002 to reveal the internal support cap 6010 that caps the pellet collection reservoir (pellet collection reservoir 6014 in FIG. 6D). In addition to the external protective cap 6004, the internal support cap 6010 can protect the distal end 6005 of the tubular body 6002 and prevent leakage from the tube upon storage or use, for example, particularly during centrifugation. In some embodiments, the support cap 6010 can be removed. Removal of the distal protective cap 6004 also reveals the support ribs 6012 that can be included to reinforce and protect the distal end 6005 of the tubular body 6002, particularly during centrifugation. The support ribs 6012 can be removed in some embodiments. For example, a specially designed centrifuge bucket insert may be designed to support a distal portion of the tubular body 6002, perhaps with the support ribs 6012 eliminated.

[0217] FIG. 6C shows a view of the centrifugal concentrator 5010 similar to FIG. 6B, except with the closure cap 6006 removed to illustrate how the closure cap 6006 is attached to the tubular body 6002. In an exemplary embodiment, the proximal end 6001 of the tubular body 6002 includes threads 6011 that allow the closure cap 6006 to be threadedly attached to the tubular body 6002. However, the threads 6011 are merely exemplary. The threads 6011 may be replaced with any structure known in the art to perform the same or a similar function. For example, the closure cap 6006 may be sealed to the tubular body 6002 by a bayonet mount, a friction arrangement, an o-ring assembly, or the like, on the tubular body 6002 or on the cap 6006.

[0218] 6D-6F, there is shown a detail of the distal end 6005 of the tubular body 6002 and how the plunger 6008 can eject the microbial pellet. In an exemplary embodiment, the distal end 6005 of the tubular body 6002 includes a pellet collection reservoir 6014. For reference, the pellet collection reservoir 6014 is shown covered by a support cap 6010 in FIGS. 6B and 6C. As discussed in more detail elsewhere herein, in some embodiments, the centrifugal concentrator 5010 is designed to be centrifuged in a swinging bucket centrifuge such that the microorganisms are pelleted at the bottom of the tube (as opposed to on the sidewall, which is typical for fixed angle centrifuge rotors). Thus, substantially all of the unlysed microorganisms in the sample should be able to pellet in the pellet collection reservoir 6014. In one embodiment, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of the microorganisms in the sample are pelletable in the pellet collection reservoir 6014. In one embodiment, the pellet collection reservoir 6014 may be sized and designed to contain substantially all of the microorganism pellets (e.g., about 500 μl, about 400 μl, about 300 μl, about 200 μl, 20-200 μl, 40-150 μl, or about 50-100 μl or less). In some embodiments, the tubular body 6002 may include a sloped interior sidewall 6015 designed to funnel the microorganisms into the pellet collection reservoir 6014 (see FIGS. 6E and 6F).

[0219] In some embodiments, the pellet collection reservoir 6014 may include a breakaway end 6016 designed to allow a portion of the plunger 6008 to be forced through the pellet collection reservoir 6014 to expel the microbial pellet. Suitable examples of a breakaway end include, but are not limited to, a thinner molded part, a thinner molded part with a frangible region, a foil cap, and the like.

[0220] 6E and 6F, there is shown a detail of how the plunger 6008 penetrates the pellet reservoir 6014 and expels the microbial pellet from the distal end 6005 of the tubular body 6002. The plunger 6008 and pellet reservoir 6014 are designed such that the microbial pellet can be expelled from the pellet reservoir while isolating the spent lysate in the tubular body. The plunger 6008 includes a distal portion 6030 having a tip 6022 designed to collect the microbial pellet and to penetrate the end 6016 of the pellet reservoir 6014 (e.g., a fixed foil cap or a frangible separation portion). In an exemplary embodiment, the tip 6022 is a shovel in the shape of a sharp edge 6023 that can penetrate the end 6016 of the pellet reservoir 6014. While the tip 6022 is shown in an exemplary embodiment as a shovel shape, other suitable shapes include, but are not limited to, a blade shape, a blunt end, a spike end, and the like. When the end of the pellet reservoir 6014 is pierced and the microbial pellet is discharged, it is desirable for the spent isolate to remain within the tubing body so that the spent lysate does not leak out and dilute the pellet. In some embodiments, the spent lysate may contain potentially infectious or biohazardous materials, and for that reason containing the spent lysate is an important safety feature. Near the distal end 6030 of the plunger 6008, in one embodiment, there is a portion 6018 that is sized and designed to connect with an interior portion 6020 of the pellet reservoir 6014. In this embodiment, the interface between the portion 6018 of the plunger 6008 and the interior portion 6020 of the pellet reservoir 6014 creates a seal that isolates the spent lysate in the tubing body 6002. The interface may also ensure that the microbial pellet is efficiently collected and discharged. While the interface between 6018 and 6020 is shown as a friction fit, either portion 6018 or portion 6020 may include, for example, an O-ring or similar structure to create a seal that isolates the spent lysate in tubular body 6002.In some embodiments, actuation of the plunger 6008 expels the pellet from the distal end of the centrifugal concentrator under pressure by opening the separation end 6016. In this embodiment, as the plunger is depressed, the interface between portion 6018 and the interior portion 6020 of the pellet reservoir 6014 causes an increase in pressure within the cavity until the separation end 6016 is pierced, resulting in the microbial pellet being expelled from the pellet reservoir 6014. This may result in a greater recovery of microorganisms since there is less opportunity for the microorganisms to become retained on the sides of the pellet reservoir due to pressure.

[0221] 6G, the plunger 6008 is shown alone. The plunger 6008 includes a proximal end 6024, a distal end 6030, a retaining member 6026, a portion 6018, and a shovel tip 6022, which are discussed elsewhere herein. The plunger 6008 includes a plunger shaft 6028, which in one embodiment is sized such that when the plunger is depressed, it can penetrate the end of the tubular body 6002 and expel the microbial pellet (e.g., be substantially the same length as the tubular body 6002). Additionally, the shaft may optionally include an o-ring 6027 or similar structure that may be designed to connect with the cap 6006 and seal the interface between the cap and the plunger. Of course, the cap may also include a sealing member instead of or in addition to the o-ring 6027. It is understood that the centrifugal concentrator 5010 is merely exemplary. The centrifugal concentrator 5010 and variations thereof discussed above, as well as other vessels, may be used with the various methods disclosed herein.

[0222] Investigation steps Once the microorganisms have been pelleted, the pellet can be interrogated and the microorganisms in the pellet can be identified and / or characterized. In one embodiment, the interrogation may be performed in a non-invasive manner, i.e., while the pellet is interrogated, it remains in the centrifugal concentrator. The ability to identify microorganisms in a non-invasive manner, optionally in conjunction with maintaining the hermeticity of the container throughout the separation and identification process and automating some or all of the procedure, avoids constant handling of contaminated and / or infectious samples, greatly increasing the safety of the overall process.

[0223] In another embodiment, the pellet can be interrogated using molecular techniques (e.g., PCR) to amplify sequences of microbial RNA or DNA that can be used to individually identify each of the types of microorganisms that may be present in the sample. In one example, nucleic acid sequences characteristic of each of the individual types of bacteria, fungi, etc. that may be present in the sample can be selected, forward and reverse primers can be designed for amplifying those sequences, the microorganisms in the pellet can be lysed, and the lysate (or nucleic acid purified from the lysate) can be combined with primers and other PCR reagents (buffers, polymerase, etc.), whereby the selected characteristic nucleic acid sequences can be amplified according to procedures well known in the art. The amplified nucleic acid can be detected and used to identify the presence of one or more microorganisms in the sample according to procedures well known in the art, including, but not limited to, real-time detection or post-amplification analysis such as melting curve analysis, fluorescently labeled, radioactively labeled, chemiluminescently labeled, enzyme-labeled other dsDNA binding dye techniques and probes, as known in the art. In one embodiment, the pellet can be interrogated using the FilmArray system detailed elsewhere herein. In one embodiment, the pellets can be interrogated using a specially adapted blood culture identification (BCID) panel pouch and protocol. BCID panels and protocols are described in U.S. Patent No. 10,053,726, which is incorporated herein by reference in its entirety. However, BCID is merely one example of an assay device. Those skilled in the art will appreciate that the pellets can be interrogated using a specially designed assay, preferably with a sensitivity and detection limit that is suitably adapted to the concentration of organisms in a sample obtained, for example, directly from blood.

[0224] In another embodiment, the pellets can be investigated by sequencing the nucleic acids present in the pellets. Sequencing of characteristic microbial sequences or entire microbial genomes can be used to identify the microorganisms in the pellets. Such sequencing may be performed according to one or more of the many sequencing techniques known in the art. In one embodiment, the sequencing is Sanger sequencing. In another preferred embodiment, the sequencing comprises massively parallel or "next generation" sequencing (NGS) techniques. In massively parallel / NGS techniques, hundreds of thousands to millions of DNA fragments are processed in parallel, resulting in low cost per base of sequence generated and throughput on the gigabase (Gb) to terabase (Tb) scale in a single sequencing run. As a result, massively parallel / NGS techniques can be used to characterize entire genomes at low cost and high throughput. EXAMPLES

[0225] Example 1 - Detection of Microorganisms Directly from Whole Blood The differential lysis buffer and centrifugal concentrator described herein can be used for the detection of microorganisms directly from whole blood. For example, they can be used to rapidly identify microorganisms that cause sepsis without pre-culturing the blood sample and amplifying the disease-causing organisms prior to detection. However, as discussed elsewhere herein, such detection and diagnosis directly from whole blood has proven difficult for several reasons. For one, the number of infectious organisms found in whole blood in BSIs is usually low (approximately 1-100 colony forming units (cfu / ml) per mL of blood, with approximately 1-10 cfu / ml being typical in most individuals with culture-confirmed sepsis), and blood contains several inhibitors of polymerase chain reaction (PCR) (e.g., hemoglobin and leukocyte-derived genomic DNA, which co-purify with the microorganism and can interfere with both nucleic acid recovery from the target microorganism and downstream PCR).

[0226] Due to the presence of very few organisms and PCR inhibitors in whole blood, enrichment from larger volumes of whole blood (e.g., 1-20 mL) is desirable to obtain the quality and quantity of DNA template desired to achieve sensitivity at clinically relevant microbial levels. In one embodiment, the differential lysis buffer and centrifugal concentrator described herein allows a technician to lyse non-microbial cells in about 1-20 mL (e.g., about 10 ml) of whole blood and concentrate the microorganisms therein by centrifugation within about 5-20 minutes (e.g., about 15 minutes). Illustratively, the lysis of the sample does not include a DNase step, and the centrifugal concentrator does not use a high density cushion. This makes sample preparation faster, easier, and more reproducible.

[0227] The microbial pellet obtained from the centrifugal concentrator described herein can be discharged directly into a sample vial that can be used to inject the sample into a molecular assay device. In a specific example, the microbial pellet obtained from the centrifugal concentrator can be discharged directly into a FilmArray Injection Vial (FAIV) (described in U.S. Pat. No. 10,464,060, incorporated herein by reference in its entirety) and then into a FilmArray pouch. In one embodiment, the FAIV can be used to directly inject the microorganism obtained from the centrifugal concentrator into a blood culture identification (BCID) panel pouch for identification of the microorganism in the sample. Currently, the BCID panel assay takes about 60 minutes to perform. The BCID panel assay and protocol are described in U.S. Pat. No. 10,053,726, incorporated in its entirety as above. The inventors in this case have used differential lysis buffer and centrifugal concentrators in conjunction with the BCID panel assay and specially modified device protocols to achieve a detection limit of about 1-10 cfu / ml, and an overall sample preparation and analysis time of about 75 minutes. However, further improvements in chemistry and instrument performance will likely result in significant reductions in analysis times.

[0228]

[0033] Referring now to Figure 7, an example sample preparation workflow is illustrated. The first step 700 is to provide a blood sample and a centrifugal concentrator. In one example, the blood sample, which may have a volume of about 10 ml, is provided in a standard vacutainer. In one example, the centrifugal concentrator may provide a volume (e.g., about 30 ml) of differential lysis buffer therein.

[0229] In a second step 702, the blood sample and the differential lysis buffer of Table 1 are combined for a time sufficient to lyse substantially all of the non-microbial cells in the sample (i.e., all of the blood cells) and obtain a lysate. For example, the blood sample and the differential lysis buffer may be combined for about 1-5 minutes, although longer or shorter times may be used. Illustratively, lysis may be performed at a temperature of about 2° C. to about 45° C., e.g., about 15° C. to about 40° C., e.g., about 30° C. to about 40° C. In one embodiment, lysis may be performed at room temperature.

[0230] After combining the blood sample with the differential lysis buffer for a sufficient time to obtain a lysate, in step 704, microorganisms, if present, may be recovered from the lysate by centrifugation. In one embodiment of the invention, the centrifugal concentrator may be centrifuged in a swinging bucket rotor such that the microorganisms form a pellet directly on the side of the tube. The vessel is centrifuged at a sufficient acceleration and for a sufficient time such that the microorganisms are pelleted and / or separated from other components of the sample. In one embodiment, the centrifugation time may be from about 30 seconds to about 30 minutes, e.g., about 10 to 15 minutes. Illustratively, the centrifugal acceleration may be from about 1,000×g to about 20,000×g, e.g., about 3000 to 10,000×g. Illustratively, the centrifugation may be performed at a temperature of from about 2° C. to about 45° C., e.g., about 4 to 8° C.

[0231] If the centrifugal concentrator includes an optional distal support cap, the support cap may be removed prior to plunging, as illustrated in step 706. Steps 708-712 illustrate an example process for expelling a microbial pellet from a centrifugal concentrator. In step 708, at the start of the plunge, the distal end of the plunger moves into the pellet reservoir and the pellet can be isolated from the supernatant. This was illustrated in FIG. 6E, previously discussed herein. As the plunger is pushed further into the pellet reservoir, the isolated end of the pellet reservoir may be punctured, separated, crushed, etc., and the pellet may begin to be expelled, as illustrated in step 710. Step 712 in the workflow shows the pellet being completely expelled from the centrifugal concentrator. The expelled pellet can be used in a number of assays known in the art to characterize and identify the microorganisms in the pellet. As previously discussed herein, PCR analysis and sequencing are two non-limiting examples of assays that can be used to characterize and identify the microorganisms in the pellet.

[0232] As shown at 714, in one embodiment, the distal end of the centrifugal concentrator may be sized and designed to fit directly into a container. In one embodiment, the container is a FilmArray Injection Vial (FAIV). As such, the pellet may be directly ejected into the FAIV for characterization and identification of the microorganisms in the pellet; the FAIV may then be used to inject the sample into a FilmArray assay pouch. In one embodiment, the pellet may be ejected directly into the FAIV, and the FAIV may be used to inject the microorganisms into the FilmArray pouch without moving the FAIV away from the centrifugal concentrator. After the FAIV is used to load the sample into the FilmArray pouch, the entire assembly may be disposed of in a biohazard waste container. This reduces the handling of potentially infectious organisms and potentially biohazardous waste, limiting the risk of contamination.

[0233] In one embodiment, an aliquot (e.g., about 1 ml) of the original sample may be added to the pellet discharged in step 712, to a vial in step 714 together with the pellet, or directly to the analyzer together with the pellet. The lysis and centrifugation methods (and related methods) described herein are well suited for concentrating and detecting microorganisms such as bacteria and yeast, but are not particularly suited for detecting viruses. Viruses can also be isolated and detected by adding an aliquot of the original sample to the pellet and then to the analysis.

[0234] In one aspect of the invention, some or all of the method steps can be automated. Automating the method steps allows for more efficient testing of a larger number of samples and reduces the risk of human error in handling samples that may contain harmful and / or infectious microorganisms. But more importantly, automation allows for definitive results to be obtained without delay at any time of the day or night. Several studies have shown that more rapid identification of sepsis-causing organisms correlates with improved patient care, shorter hospital stays, and lower overall costs.

[0235] Referring now to FIG. 8, the sample preparation time of the method described herein using differential lysis buffer and centrifugation is compared to other methods. As is evident from FIG. 8, the method of combining the sample with differential lysis buffer followed by centrifugation involves only two steps and a total processing time of about 15 minutes. This is significantly faster and easier than other methods tested by the inventors in this case. The MolYsis method discussed in the introductory section of this document involves several complex steps, including a DNase step, and takes about 45-50 minutes just for eukaryotic cell lysis, DNase treatment, and microbial cell recovery. Washing and lysis of microbial cells requires additional steps and buffers. Successful use of the MolYsis system requires skilled professionals. Dependence on operator skill creates the risk of inter-operator variability in the yield and quality of the results. Many buffers and manual pipetting steps increase the risk of cross-contamination of samples. Combining the sample with differential lysis buffer followed by centrifugation does not require many of those steps, including the DNase step, nor does it require many of the washing steps. In the methods described herein, the microbial cells recovered after centrifugation are suitable for post-centrifugation molecular analysis (eg, PCR assays, DNA sequencing, or mass spectrometry) without further processing.

[0236] FIG. 8 also compares the sample preparation time of the differential lysis and centrifugation method with two other protocols. The Y2 protocol is a procedure using saponin-based lysis combined with protease and DNase digestion steps. The Y2 protocol required several complicated steps and about 90 minutes for sample preparation. The Lycoll+DNase protocol is a procedure using saponin-based lysis combined with Ficoll density gradient in DNase digestion and centrifugation. The Lycoll procedure resulted in good organism yields and effectively removed genomic DNA, but included complicated layering of the lysate on the Ficoll density gradient and a processing time of about 2 hours. Compared to the method claimed herein, which uses differential lysis buffer and subsequent centrifugation, both the Y2 protocol and the Lycoll+DNase procedure include complicated steps and excessive time. The same applies to the comparison of the differential lysis and centrifugation method with the MolYsis method.

[0237] FIG. 9 illustrates the microbial recovery in one set of experiments that can be achieved by treating samples with differential lysis buffer and subsequent centrifugation. The control is spiked buffer and the test sample is spiked whole blood. The control and test samples were spiked with the same number of organisms. The control tests the ability to recover spiked organisms from the buffer by centrifugation, while the test samples demonstrate the effectiveness of the differential lysis buffer in lysing eukaryotic cells (i.e., RBCs, white blood cells, platelets, etc.) and recovering the spiked microbial cells from the lysate by centrifugation. Compared to the control, in this experiment, approximately 86% of the organisms can be recovered from the whole blood sample by the method that includes treating the spiked blood sample with differential lysis buffer and subsequent centrifugation. As illustrated in Table 2, the recovery rate can exceed 90% in other experiments.

[0238] [Table 2]

[0239] In preferred embodiments, the recovery of microorganisms by differential lysis followed by centrifugation may be at least 85%, at least 90%, at least 95%, at least 99% or 100%.

[0240] Also, genomic DNA was efficiently removed by treating blood samples with differential lysis buffer followed by centrifugation, all without a DNase step or other complex or time-consuming processing steps. Table 3 below compares the extent of genomic DNA removal from whole blood achieved with differential lysis buffer and centrifugation versus the Lycoll and Lycoll+DNase methods. The whole blood control represents the amount of genomic DNA recovered from the lysed whole blood sample. DNA was purified with the MagnaPure system and quantified using the ThermoFisher Quantifiler Human DNA Quantification Kit.

[0241] [Table 3]

[0242] As is evident from Table 3, the amount of genomic DNA recovered with the differential lysis buffer and centrifugation process is significantly better than the Lycoll method and comparable to the Lycoll+DNase method. The differential lysis buffer and centrifugation process is significantly faster, easier, and more reproducible than either the Lycoll or Lycoll+DNase methods, and achieves impressive genomic DNA reduction without the time-consuming DNase step.

[0243] This is demonstrated in a slightly different form in FIG. 10, which compares the crossing point (Cp) values ​​for amplification of various amounts of yeast control in the presence of whole blood material pelletable by centrifugation after treatment of the blood with differential lysis buffer. In this case, the differential lysis buffer (alkaline) was used in the Lycoll method ( *For the differential lysis buffer experiments, 10 mL of whole blood was spiked with 1, 10, or 100 CFU / mL of yeast control, then processed with differential lysis buffer and subsequent centrifugation as described herein. The resulting pellet was transferred to a FAIV, and samples were run in a FilmArray BCID pouch. Lycoll experiments were run similarly, except that 10 mL of spiked whole blood was processed using the Lycoll method. CFU controls (■) represent Cp in the absence of any whole blood material in various amounts of yeast DNA. CFU amounts of different yeast were diluted in PBS and pipetted into FAIV at levels equal to 100% concentration of organisms from the 10 mL of spiked whole blood used for the differential lysis buffer and Lycoll procedures (1 CFU / mL in whole blood = 10 CFU control into FAIV). WB control (□) is unconcentrated whole blood. For the WB control, 200 μL of spiked whole blood was pipetted into the FAIV at a level equal to 100% concentration of organisms from the 10 mL spiked whole blood used in the differential lysis buffer and Lycoll procedure, giving an initial LoD / Cp value of organisms, followed by the enrichment protocol. As can be seen in FIG. 10, the amplification of yeast DNA in the presence of the Lycoll pellet was delayed by approximately 3 Cp units compared to the CFU and WB control. In contrast, no detectable inhibition was observed from the pellet obtained using differential lysis buffer and subsequent centrifugation. That is, the amplification of yeast DNA in the presence of the pellet obtained from the differential lysis buffer is virtually indistinguishable from the CFU and WB control. The increase in Cp in the Lycoll pellet appears to be due to the high levels of hgDNA that are enriched in the pellet along with the spiked yeast organisms. hgDNA is a known competitive inhibitor of DNA recovery with magnetic silica beads and a non-specific inhibitor of PCR. Based on the data shown in Table 3, and based on these data, it concluded that the pellet obtained from the differential lysis buffer does not have as much hgDNA in the pellet, and as a result has a more similar Cp of yeast DNA to the whole blood control, or even to the CFU control with no matrix at all.

[0244] Referring now to FIG. 11, data is presented for different differential lysis buffer formulations (LB18-LB21) with various amounts of CAPS and BrijO10. For lysis buffer testing, 10 ml whole blood samples were spiked with E. Coli, Enterobacteriaceae, or Yeast and the samples were processed using the indicated differential lysis buffer and centrifugation process according to the methods described herein. The resulting pellet was transferred to a FAIV and the samples were run on a FilmArray BCID pouch as described in FIG. 10. WB controls were the same as those described in FIG. 10. The data presented in FIG. 11 shows that the differential lysis buffer efficiently lyses host cells (i.e., RBCs, white blood cells, platelets, etc.) and host cell nuclei while leaving microbial cells intact and pelletable by centrifugation.

[0245] Using the differential lysis buffer disclosed herein, sample processing involves only two simple steps, and processing time can be reduced to about 15 minutes. The DNase step, which is common in other methods, may be eliminated due to the effective disruption of the nuclear membrane. The volume of the pellet obtained by the differential lysis buffer and subsequent centrifugation may preferably be <200 μL. BrijO10 and CAPS completely lysed blood cells within seconds or minutes. As shown in Example 1, this buffer is easy to handle, and therefore the results should be more reproducible (Figure 8). It can rapidly enrich microbial cells from whole blood (Figure 8), recover a high percentage of microbial cells in the sample (Figure 9 and Table 2), reduce human genomic DNA from the microbial pellet (Figure 10 and Table 3), and effectively lyse host cells and their nuclei while leaving the microbial cells intact and recoverable by centrifugation (Figure 11).

[0246] Example 2 - Microbial recovery by species at low spike levels (<1 CFU / mL) The previous examples have shown that the differential lysis buffer and centrifugal concentrator described herein can be used to lyse total blood factors, recover microbial cells, and then detect the microorganisms. This example expands on Example 1 and demonstrates the ability to recover and identify microorganisms at low levels (i.e., <1 CFU / mL) from spiked whole blood. In most cases of bloodstream infection (i.e., sepsis), clinically relevant levels of microorganisms in whole blood range from <about 1 CFU / mL up to about 10 CFU / mL. This example also demonstrates the ability to recover and identify microorganisms at clinically relevant levels on a species basis.

[0247] A direct result of the blood processing method described herein is an uncomplicated workflow with steps of lysing, centrifuging, and draining the pellet to recover the organisms from the lysate. In this example, the whole blood sample was mixed with differential lysis buffer, lysis was allowed to proceed for about 5 minutes, and the lysate was centrifuged at about 3000×g for about 30 minutes to recover the organisms. The lysis buffer used in this example is shown in Table 4.

[0248] [Table 4]

[0249] The comprehensive test panel included 120 organism strains from 12 species of bacteria and yeasts most commonly isolated from bloodstream infections. The collected organisms not only maintained viability but also had reduced levels of blood debris and contaminating host DNA, facilitating their potential use as inputs into a variety of downstream applications, both growth-based and molecular analyses.

[0250] FIG. 12 illustrates the workflow used in this study. In steps (a) and (b), a biological stock of appropriate concentration (e.g., about 100 CFU / ml) can be obtained by serially diluting and plating the biological stock until the desired concentration is obtained. The concentration can be verified by plating the stock on an agar plate and growing individual colonies on the plate. For example, if plating 50 μL of a 100 CFU / ml stock, 5 colonies / plate should be obtained. The stock biological solution can be diluted and plated several times to obtain the desired concentration. In step (c), the stock biological solution (e.g., about 100 CFU / mL) was spiked into whole blood. In the example illustrated in FIG. 12, 150 μL of biological stock was spiked into 30 mL of whole blood. It was desired to spike the organisms at a concentration of <1 CFU / mL. In the example shown in FIG. 12, the target spike concentration was 0.5 CFU / mL. As explained in more detail below, spikes were varied between gram-negative, gram-positive, and yeast organisms. The spiked whole blood was divided into three 10 mL fractions and combined with 20 mL of LB100 buffer in a centrifugal concentrator and allowed to lyse for 5 min at room temperature, steps (d)-(f). In the specific example illustrated in Figure 12, the blood and lysis buffer were inverted 10 times in the tube of the centrifugal concentrator, incubated at RT for 5 min, vortexed for approximately 5 s, and then centrifuged in a swinging bucket centrifuge rotor at 3000 x g for 30 min.

[0251] After centrifugation, the pellet from the centrifugal concentrator was drained into 500 μL of TSB (tryptic soy broth) (step (g)) and 100 μL was plated onto each of five agar plates (step (h)). The plates were incubated at 37° C. for 24 hours (step (i)), and the CFU from the five plates were added to obtain the total recovery (step (j)). While in this example plating and culturing was used for the detection of the organisms, the workflow could be used for a variety of different types of detection. For example, molecular detection techniques such as, but not limited to, PCR (e.g., by the FilmArray system, as discussed in detail herein), whole genome sequencing, or molecular AST could be used. Phenotypic (e.g., Vitek2 AST), proteomic (e.g., maldi-TOF, Vitek MS, etc.), and microscopy techniques could be used to investigate the pellet obtained from the centrifugal concentrator.

[0252] The results of this research study are summarized below.

[0253] The percent recoveries for all organisms, Gram-negative, Gram-positive, and yeast, are shown below in Table 5. The average overall recovery in this study was 80%, which exceeded the target goal of >70%.

[0254] [Table 5]

[0255] FIG. 13 illustrates that some variability was observed among the organism recoveries, but all organisms could be recovered and cultured. As is evident in FIG. 13, recovery of S. pneumoniae strains was low in this study. Recovery of Gram-positive species increases to 95% when S. pneumoniae strains are excluded, with overall recovery increasing to 84%. Further method optimization, e.g., pH, contact time, supplementation, may improve recovery of this species. It is also possible that S. pneumoniae strains are less viable (compared to other organisms) after recovery from the lysis buffer, and their apparently low relative recoveries would increase if detection techniques not dependent on viable organisms (e.g., molecular techniques) were used for detection.

[0256] The percent recoveries by CFU for total organisms, gram negative, gram positive, and yeast are shown in Table 6 below.

[0257] [Table 6]

[0258] The measured input inoculum levels for all organisms were somewhat higher than the target of 5 CFU / 10 mL. Nevertheless, all of the goals of achieving recovery and detection of <1 CFU / mL from whole blood were met. Figure 14 expands on the data from Table 6 on a per organism basis.

[0259] Table 7 (below) shows the reduction of contaminating host DNA.

[0260] [Table 7]

[0261] An average reduction of 99.7% of host DNA was calculated from the input DNA concentration of 10 mL of blood in the lysate relative to the host DNA in the expelled pellet. The range shown reflects the variation across 15 different blood donors and test dates.

[0262] For a comprehensive organism panel, the test demonstrated recovery at low spike levels (i.e., <1 CFU / mL) from whole blood. Recovery and detection sensitivity in this test is comparable to that of conventional blood cultures (4-8 CFU / 10 mL). CAPS-Brij lysis buffer lyses and solubilizes human blood cell membranes, RBCs, and WBCs. CAPS-Brij lysis buffer also reduced blood cell debris and DNA in the discharge pellet. The processing method enriches and collects viable organisms, significantly reduces levels of blood debris and host DNA, and provides a sample potentially suitable for multiple rapid diagnostic pathways.

[0263] Example 3 - Cultivation of microbial cells after lysis and collection of whole blood In this example, different differential lysis buffer compositions were compared for detection in the FilmArray BCID assay pouch. Only three organisms were used in this comparison: C. albicans, E. Coli, and S. agalactiae. ACD (anticoagulant acid citrate dextrose) anticoagulated blood was used in this study. This study demonstrates that (1) the differential lysis buffer / centrifugation procedure described herein can enrich cells and test all buffer compositions and organisms, and (2) culture after centrifugation can enrich cells of organisms isolated from blood and lysis buffer and test all selective lysis buffer compositions. All selective lysis buffers tested are capable of lysing blood cells while leaving microbial cells intact and viable.

[0264] The buffers tested are listed in Table 8 below.

[0265] [Table 8]

[0266] LB20 is a buffer listed in Table 1 and is the buffer used in the studies described in Example 2.

[0267] The data in Table 9 show an improvement in the crossing point (Cp) of the alkaline lysis / centrifugation method versus non-concentrated spiked whole blood. The improvement in Cp is likely due to the removal of PCR-interfering substances (e.g., hemoglobin) and the concentration of cells in the sample.

[0268] [Table 9]

[0269] For buffers LB16, LB19, and LB20, the apparent enrichment was about 8-fold, and in LB100, it was about 2.5-fold. A one-cycle improvement in Cp represents about a 2-fold increase in the input concentration of target cells or template DNA, a two-cycle improvement in Cp represents about a 4-fold increase, a three-cycle improvement in Cp represents about an 8-fold increase, and so on (according to the general formula that an n-cycle improvement in Cp represents about a 2n-fold increase in the input concentration of target cells or template DNA).

[0270] The data in Table 10 shows the improvement in Cp resulting from 3 hours of incubation in medium of the pellet collected from the centrifugal concentrator. 150 uL of BHI broth was mixed with the pellet from the centrifugal concentrator and incubated at 37° C. for 0 hours or 3 hours. The Cp improvement shown in Table 10 represents the average reduction in Cp (i.e., reduction in time to detection) observed in the 3 hour incubation relative to the sample incubated for 0 hours.

[0271] [Table 10]

[0272] Incubation of the cells for 3 hours enriched the cells from LB16 approximately 2-fold, approximately 12-fold in the case of LB19 and LB20, and approximately 8-fold in the case of LB100.

[0273] Figure 22 illustrates another experiment comparing culture after lysis and centrifugation on organisms recovered from ACD and SPS anticoagulant blood. The test was performed on C. albicans, E. Coli, K. pneumoniae, S. agalactiae, and S. aureus. After lysis in LB20 and recovery of cells by centrifugation, 150 uL of BHI broth was mixed with the pellet from the centrifugal concentrator and incubated at 37°C for 0 or 3 hours. The Cp improvement shown in Figure 22 represents the average reduction in Cp (i.e., reduced time to detection) observed in the 3 hour incubation relative to the 0 hour incubation sample.

[0274] In this study, cells from ACD blood showed an improvement in Cp of about 5.5 after 3 hours or incubation at 37°C, and cells recovered from SPS blood showed an improvement in Cp of about 3 after 3 hours or incubation at 37°C. The improvement in E. Coli and S. aureus was the most dramatic. C. albicans, which grows more slowly than the bacteria, improved by about 1 Cp in ACD blood and actually performed somewhat worse in SPS blood. No ACD performance data was available for K. pneumoniae in this study. This study illustrates that certain anticoagulants can affect growth and culturability in some organisms. For all organisms in this study, ACD appeared to be less detrimental to growth and culturability compared to SPS.

[0275] Example 3 - Flow-through lysis, incubation, and volume reduction system In addition to or in combination with other devices discussed herein, flow-through systems can be used for cell lysis, incubation, and volume reduction. A schematic diagram of one example of such a system 1500 is illustrated in Figure 15. The flow-through system 1500 includes three adjacent buffer chambers 1502, 1506, and 1510, each containing a first buffer 1504, a second buffer 1508, and a third buffer 1512, respectively. System 1500 further includes a channel 1514 (e.g., a tube or open trough of buffer exchange membrane in contact with buffers 1504, 1508, and 1512 in buffer chambers 1502, 1506, and 1510, respectively. In one embodiment, first buffer 1504, second buffer 1508, and third buffer 1512 may be comprised of a selective lysis buffer for culturing microbial cells, a culture medium or nutrient broth (e.g., a nutrient broth for culturing bacterial organisms, a fungal organism, or a culture medium suitable for culturing both bacterial and fungal organisms), and a hypertonic solution / media for reducing sample volume. In one embodiment, system 1500 also includes a buffer exchange membrane in contact with buffers 1504, 1508, and 1512 in buffer chambers 1502, 1506, and 1510, respectively. In one embodiment, first buffer 1504, second buffer 1508, and third buffer 1512 may be comprised of a selective lysis buffer for culturing microbial cells, a culture medium or nutrient broth for culturing bacterial organisms, a culture medium suitable for culturing both bacterial and fungal organisms, and a hypertonic solution / media for reducing sample volume. To enhance the lysis and volume reduction, the system may include a temperature control system (not shown) that can adjust and control the temperatures of the first buffer 1504, the second buffer 1508, and the third buffer 1512 (individually or as a group). For example, the selective lysis may be performed at room temperature, the microbial culture may be performed at 32-37° C., and the volume reduction may be performed at 4° C. A sample (e.g., a whole blood sample) disposed within the channel 1514 may be selectively exposed to each of the buffers 1504, 1508, and 1512 in any given order, or to two or more buffer chambers simultaneously, to achieve, for example, blood cell lysis, microbial cell culture, and sample volume reduction / concentration.

[0276] In one embodiment, a whole blood sample containing microbial cells (e.g., a whole blood sample from a subject suspected of having sepsis) may be added to channel 1514, thereby selectively exposing the blood sample to each of buffers 1504, 1508, and 1512. Buffer exchange membranes are well known in the art. A suitable buffer exchange membrane may be selected to allow blood cell debris, hemoglobin, and other products of blood cell lysis to diffuse through the membrane while retaining microbial cells. For example, the buffer exchange membrane may be a dialysis membrane. Dialysis membranes are fabricated and characterized to have different molecular weight cut-offs (MWCOs), for example, ranging from 1 kilodalton (kDa) to about 1 MDa (i.e., 1 megadalton, or about 1,000,000 Da). The determination of the MWCO is a result of the number and average size of holes made during the fabrication of the dialysis membrane. MWCO typically refers to the minimum average molecular weight of a standard molecule that does not effectively diffuse through the membrane during extended dialysis. However, it is important to recognize that the MWCO of a membrane is not a well-defined value. Molecules with a mass close to the MWCO of a membrane will diffuse through the membrane slower than molecules significantly smaller than the MWCO. For a molecule to diffuse through a membrane rapidly, it typically needs to be at least 20-50 times smaller than the membrane's MWCO rating. Dialysis tubing in laboratory use is typically made of films of regenerated cellulose or cellulose esters. However, dialysis membranes made of polysulfone, polyethersulfone (PES), etched polycarbonate, or collagen are also widely used for certain medical, food, or water treatment applications.

[0277] Because microbial cells are relatively large and cellular debris is relatively small, channel 1514 may also be fabricated from a filtration membrane material. Membrane materials designed to filter bacteria and larger cells from solution are well known in the art. For example, a filtration membrane with a nominal pore size of 0.25 to 1 μm (e.g., 0.5 μm) may be used to retain microbial cells within channel 1514 while allowing the sample in channel 1514 to be rapidly exchanged with buffers 1504, 1508, and 1512.

[0278] With reference to FIG. 15, a sample (e.g., a whole blood sample) placed in channel 1514 may first be exposed to buffer 1504 in chamber 1502, as shown at 1516 in FIG. 15A. The sample may then be moved to be exposed to buffer 1508 in chamber 1506, as shown at 1518 in FIG. 15B, and then the sample may be moved to be exposed to buffer 1512 in chamber 1510, as shown at 1520 in FIG. 15C. While FIGS. 15A-15C show the sample being moved from one buffer chamber to the other chronologically, exposing the sample to each buffer in turn, it will be understood that the sample may be moved back and forth, e.g., exposed to a buffer more than once, exposed to two or more buffers simultaneously. Similarly, buffers 1504, 1508, and 1512 may be prepared in any given order. Table 11 illustrates some of the options.

[0279] [Table 11]

[0280] And while this example discusses microbial cell culture and sample volume reduction using selective lysis buffers, media, and hypertonic solutions for lysis, respectively, one of skill in the art will understand that these buffers are merely exemplary and that other buffers may be used with system 1500. Similarly, while system 1500 includes three buffer tanks, this is merely exemplary. Alternative versions of system 1500 may include more or less buffers. Additionally, while channel 1514 is shown as a linear channel, this is merely exemplary. Channel 1514 may include circuitous flow paths or other modifications known in the art, for example, to maximize the surface area of ​​the sample exposed to the buffer.

[0281] In one embodiment, the hypertonic medium may be sufficient to concentrate the microorganisms in the sample and allow for identification (e.g., by PCR techniques, whole genome sequencing or molecular AST, phenotypic techniques, proteomic techniques, and microscopy techniques). In other embodiments, filtration techniques may be used for concentration / volume reduction. Filtration may be performed before or after one or more of the exposure of the sample to the selective lysis buffer, medium, and hypertonic solution. In another embodiment, centrifugation techniques may be used to concentrate the microorganisms in the sample. Centrifugation may be performed before or after one or more of the exposure of the sample to the selective lysis buffer, medium, and hypertonic solution.

[0282] Example 4 - Filtration Technique In some embodiments described herein, separation of microbial cells from their environment (e.g., separation of bacterial and / or fungal cells from a whole blood sample) can be performed by filtration. Filtration techniques may be designed to retain or pass selected cells or cell sizes. For example, blood cells (e.g., red blood cells, white blood cells, platelets, etc.) may be trapped while microbial cells may pass, microbial cells may be trapped, or a combination of filtration media may be used to selectively trap large and small cells at different stages of the filtration device. Differential filtration techniques may also be utilized to separate larger and smaller cells into different fractions. For example, filtration membranes with different nominal pore sizes may be stacked (or used in a series of separate vessels) to pass and / or capture cells having a selected size range. Flow cytometry is also a well-known technique that can sort cells by size. Cells may also be captured or concentrated by active filtration techniques. For example, most cell types have specific surface factors (e.g., proteins) that can be used in affinity purification by techniques well known in the art.

[0283] An example of a differential filtration system is illustrated in FIG. 16. A whole blood sample from a subject suspected of having sepsis may be enriched for microbial cells by first passing the whole blood sample through a larger filter having a pore size of 8-15 μm (e.g., 10 μm) to filter out larger cells such as white blood cells (WBCs) and some red blood cells. Any microbial cells will flow through the first filter. In one embodiment, a sub-soluble level of Brij detergent (e.g., <0.1%) can be used to ensure that any microbial cells attached to the outside of the WBCs are released, reducing the capture of microbial cells on the WBCs captured by the filter. Other detergents may suitably have other sub-soluble concentration levels, i.e., typically 0.1% to 1%. A second filter having a smaller pore size (e.g., 5 μm) can be used in tandem to enrich for microbial cells in the filtrate while removing more human cells. A final filter with a pore size of less than 1 μm (e.g., 0.45 μm) can be used to capture all microbial cells and significantly reduce the volume of the sample. The microbial cells concentrated on the filter can be used directly for identification and diagnosis (e.g., molecular identification by FilmArray, imaging, optical fluorescence of metabolic processes or metabolic consumption, conductivity, pH, etc.), the sample can be cultured (e.g., for 1-3 hours) to increase the number of microbial cells in the sample, or they can be subjected to alkaline lysis to further remove animal cells (e.g., human cells), centrifugation, and molecular identification as described herein.

[0284] In another embodiment, filtration can be used to recover cells after selective lysis with alkaline / Brij buffer (i.e. alkaline lysis) as described herein. However, it was found that proteinase K treatment was necessary to reduce the viscosity of the sample before filtration. In this regard, alkaline / Brij selective lysis buffer was added to whole blood and incubated for 5 minutes. After 5 minutes, 1 mL of 30 units / mL proteinase K was added and incubated at RT for about 5 minutes. The lysate could then be filtered through a 0.45 μm filter. As in the previous examples, for example, the filter-enriched microbial cells can be used directly for identification and diagnosis, or they can be cultured (e.g., for 1-3 hours) to increase the number of microbial cells in the sample.

[0285] In addition to the conventional filtration techniques mentioned above, filtration techniques using various types of structures may be used to selectively enrich specific cell types in a sample. Such filtration techniques may be used instead of conventional filtration or in combination with regular filtration to enrich or isolate microbial cells of interest from blood cells and reduce volume and inhibitors. Such enriched or isolated microbial cells may be subjected to culture calls (similar to conventional blood cultures, but likely more rapid since the microbial cells in the sample are enriched), FilmArray identification, or other investigative techniques. A further aspiration is to identify bacterial cells that are likely to contribute to making the process more economical for the customer (e.g., metabolic processes, or cheaper tests such as imaging of metabolic consumption, conductivity, or pH, optical fluorescence).

[0286] Various filtration techniques that can be used to enrich for specific cells are illustrated in Figures 17-20. Figure 17A illustrates a weir filter, Figure 17B illustrates a micropillar filter, and Figure 17C illustrates a cross-flow filter. Differential flow of larger and smaller cells around these structures can be used to separate smaller cells from larger cells. Figure 18 illustrates schematics of different types of pillar filters: (18A) polygonal, (18B) U-shaped, and (18C) butterfly-shaped micropillar shapes. Larger cells are immobilized in the trap structure while smaller cells pass through. Figure 18B also illustrates the concept that micropillars can be formed with structural features (shapes, pockets, etc.) to selectively retard the passage of certain cells through the micropillar structure.

[0287] While Fig. 17 and Fig. 18 show only one set of each of these structures, such structures (and flow directions) can be used sequentially and in combination to achieve high levels of separation. Fig. 19 and Fig. 20 illustrate this principle. Fig. 19 illustrates the separation of large and small cells in a structure with a series of micropillars and cross-flow of buffer and cell suspension. The structure of Fig. 19 separates large and small cells by deterministic lateral displacement. Large cells move away from small cells in a streamlined manner due to the modified size and spacing of the microposts in the fluid channel. Fig. 20 illustrates the concentration of large and small cells by migration along an ovoid filter unit. The filter unit achieves simultaneous separation of large cells that are larger than the gap and small cells that are smaller than the gap. The rotation along the pillars at a relatively low speed induced by the shear layer of the filtrate helps to prevent clogging of large particles. The systems in Figures 19 and 20 are examples of systems that may be used to remove lysate, exchange buffer, and add growth medium in one system. That is, lysate may be input to initiate separation, buffer may be added to output lysate, and medium may be added. In addition to or instead of the centrifugal concentrators described herein, filtration concentrators, microfluidic concentrators, dielectrophoretic concentrators, FACS (fluorescence activated cell sorting), or other similar devices may be used advantageously. The benefit of selective lysis may be that it may advantageously simplify filtration concentrator mechanisms or allow them to process larger volumes before fouling.

[0288] The workflow may include one or more of chemistry, filtration, centrifugation, and identification (e.g., but not limited to, metabolic processes, or molecular identification or another technique such as imaging of metabolic consumption, conductivity, or pH, optical fluorescence, etc.). Route 1: Selective lysis with alkaline / Brij buffer, transfer of lysate to microfluidic chip for enrichment and incubation, FilmArray for detection and identification 1. Selectively lyse human cells with alkaline / Brij selective lysis buffer 2. Concentrating for microbial cells by one or more of centrifugation, filtration devices, or microfluidic chip designs a. Sorting techniques (active (e.g., flow cytometry) or passive (weir filtration, micropillar filtration, or a combination thereof) b. Capture technology (active or passive) c. Filtration techniques (usually passive but can be active) 3. Flush with growth medium In some embodiments, centrifugation, filtration, microfluidic separation, or a combination thereof may be used to remove lysate, exchange buffer, and add growth medium in one system. b. In some embodiments, additional sensing techniques may be added for positive detection of microbial cells. i. Imaging ii. Optical fluorescence of metabolic processes or metabolic consumption iii. Conductivity iv. pH v. Microresonator vi. Dielectrophoresis vii. Capacitive Sensing viii.SPR ix.FLIR 4. Release the cells from the microfluidic device for FilmArray analysis, culture, or other confirmatory processes Pathway 2: Use microfluidic chips / selective filtration for both selection / enrichment and culturing, FilmArray, or other confirmatory processes for detection. 1. Selectively separate microbial cells from human blood cells Active selection i. Flow Cytometry (Fluorescence Activated or Optical Detection) ii. Dielectrophoretic (DEP) sorting iii. Air pressure selection b. Passive selection i. Size selection ii. Inertial Selection iii. Dielectric trap iv. Selective protein adhesion process v. Acoustic trap vi. Viscoelastic (or cell stiffness) sorting in shear gradients 2. Flush with growth medium In some embodiments, centrifugation, filtration, microfluidic separation, or a combination thereof may be used to remove lysate, exchange buffer, and add growth medium in one system. b. In some embodiments, additional sensing techniques may be added for positive detection of microbial cells. i. Imaging ii. Optical fluorescence of metabolic processes or metabolic consumption iii. Conductivity iv. pH v. Microresonator vi. Dielectrophoresis vii. Capacitive Sensing viii.SPR ix.FLIR 4. Release the cells from the microfluidic device for FilmArray analysis, culture, or other confirmatory processes

[0289] The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The described embodiments should be construed in all respects as merely illustrative and not limiting. The scope of the present invention is therefore indicated by the appended claims rather than the foregoing description. While specific embodiments and details have been included in this specification and the accompanying invention disclosure for the purpose of illustrating the present invention, it will be apparent to those skilled in the art that various modifications in the methods and apparatus disclosed herein may be made without departing from the scope of the present invention, as defined in the appended claims. All modifications that come within the meaning and range of equivalence of the claims are to be embraced within their scope.

Claims

1. 1. A method for isolating and identifying a microorganism, comprising: (a) providing a volume of a blood sample suspected of containing said microorganism; (b) mixing the blood sample with a differential lysis buffer to obtain a lysate containing lysed blood cells and unlysed microorganisms; (c) concentrating the microorganisms from the lysate; (d) adding said microorganism to a device containing one or more reagents required to identify said microorganism; (e) identifying the microorganisms present in the blood sample; and Including, wherein said microorganisms, if present, are concentrated in the range of 25-100 fold relative to the volume of said provided blood sample; wherein the microorganism, if present, has a concentration in the provided blood sample in the range of < about 1 CFU / ml to about 20 CFU / ml; the differential lysis buffer comprises a buffer, a non-ionic detergent, a salt, and a pH range of about 10-11 prior to mixing the blood sample with the differential lysis buffer, and the lysate is characterized as being about 1.5-2.5 pH units below the pH buffering range of the buffer.

2. The method of claim 1, wherein steps (a) to (e) can be completed in less than about 120 minutes, preferably less than about 90 minutes.

3. 10. The method of claim 1, wherein steps (a)-(c) can be completed within a time range of about 10 to 20 minutes.

4. 10. The method of claim 1, wherein steps (d) and (e) can be completed within a time range of less than 4 hours, preferably less than 3 hours, preferably less than 2 hours, or more preferably less than 1 hour.

5. 10. The method of claim 1, wherein the microorganism is a bacterial or fungal organism associated with a blood-borne infection.

6. 10. The method of claim 1, wherein the identification comprises one or more of a molecular test, a phenotypic test, a proteomic test, an optical test, or a culture-based test.

7. 2. The method of claim 1, wherein the identification comprises isolating one or more nucleic acids characteristic of the microorganism from the microorganism, and analyzing the one or more nucleic acids to identify the microorganism present in the blood sample.

8. 8. The method of claim 7, wherein said identifying further comprises amplifying one or more nucleic acids and then detecting said one or more amplified nucleic acids.

9. 9. The method of claim 8, wherein the detection of the one or more amplified nucleic acids comprises one or more of the use of a dsDNA binding dye, real-time PCR, a post-amplification nucleic acid melting step, a nucleic acid sequencing step, a labeled DNA binding probe, or an unlabeled probe.

10. The method of any one of claims 7 to 9, wherein the identifying step can be completed within a time range of about 5 to 75 minutes.

11. 10. The method according to any one of claims 7 to 9, further comprising carrying out a culturing step on the enriched microorganism in a medium to increase the concentration of the microorganism and then carrying out the identifying step, wherein the culturing step is carried out for 4 hours or less, 3 hours or less, or 2 hours or less, preferably 3 hours or less.

12. 2. The method of claim 1, wherein the differential lysis buffer comprises a buffer substance, a non-ionic surfactant, a salt, and a pH range of about 10 to 11 prior to mixing the blood sample with the differential lysis buffer.

13. 13. The method of claim 12, wherein the differential lysis buffer has a pH of about 7.0 to 8.0 after mixing of the blood sample and the differential lysis buffer.

14. 13. The method of claim 12, wherein the buffering substance is selected from the group consisting of CABS, CAPS, CAPSO, CHES, and combinations thereof, and the buffering substance is preferably CAPS.

15. 13. The method of claim 12, wherein the pH of the differential lysis buffer mixed with the blood sample is about 1.5 to 2.5 pH units below the pH buffering range of the buffer substance.

16. 13. The method of claim 12, wherein the non-ionic surfactant is a polyoxyethylene (POE) ether, preferably one or more of Arlasolve 200 (aka poly(oxy-1,2-ethanediyl)), Brij O10, and nonaethylene glycol monododecyl ether (aka Brij 35).

17. 13. The method of claim 12, wherein the non-ionic detergent is selected from the group consisting of Triton X-114, NP-40, Arlasolve 200, Brij O10 (also known as Brij 96 / 97), octyl β-D-glucopyranoside, saponin, nonaethylene glycol monododecyl ether (also known as Brij 35), and combinations thereof.

18. 2. The method of claim 1, wherein concentrating the microorganisms from the lysate comprises centrifugation, and wherein the concentration further comprises recovering a pellet fraction comprising the microorganisms from a supernatant fraction comprising a lysed blood fraction.

19. placing the blood sample mixed with the differential lysis buffer into a centrifugal concentrator; centrifuging the centrifugal concentrator to concentrate the microorganisms from the blood sample disposed in the chamber; allowing the concentrated microorganisms to exit a second opening at a second end of the chamber, preferably aseptically pelleting the microorganisms from the second end of the centrifugal concentrator into a vial or assay device; wherein the centrifugal concentrator further comprises: a chamber having an opening at a first end and a sealing portion at the second end, the sealing portion designed to seal the second opening at the second end of the chamber; and a plunger movably disposed at least partially inside the chamber, the plunger being designed to be actuated to open the seal portion; The method of claim 1 , comprising:

20. 20. The method of claim 19, wherein the centrifugal concentrator does not include a high density cushion or physical separator to separate the microorganisms from the lysate.

21. 20. The method of claim 19, which does not include one or more of: mixing the blood sample and the differential lysis buffer in a first container and then transferring the lysate to a centrifugal concentrator containing components other than the blood sample and the differential lysis buffer in the centrifugal concentrator; opening the centrifugal concentrator after centrifugation and decanting a supernatant fraction; an incubation step before mixing the blood sample with the differential lysis buffer; or a DNase step to digest genomic DNA in the lysate.

22. 10. The method of claim 1, which does not include one or more of an incubation step prior to mixing the blood sample with the differential lysis buffer, or a DNase step to digest genomic DNA in the lysate.

23. The method of claim 1 , wherein the microorganisms are concentrated from the lysate by a filtration technique.

24. 24. The method of claim 23, further comprising adding the filter having concentrated microorganisms thereon to one or more of a culture device or an assay device designed to identify the microorganisms present in the blood sample.

25. 2. The method of claim 1, wherein the steps of mixing the blood sample with the differential lysis buffer to obtain the lysate and separating the microorganisms from the lysate are accomplished in a single tube.

26. 26. The method of claim 25, wherein the differential lysis buffer is a single buffer provided in the single tube.

27. 26. The method of claim 25, wherein the differential lysis buffer does not contain a DNase or a protease, and the method does not include adding an exogenous DNase or protease to the single tube.

28. 2. The method of claim 1, wherein the differential lysis buffer is compatible with an anticoagulant selected from the group consisting of EDTA, citrate, acid citrate dextrose (ACD), sodium polyanethole sulfate (SPS), heparan, sodium fluoride / oxalate, and combinations thereof.

29. 1. A method for concentrating and identifying microorganisms from blood, comprising: (a) providing a blood sample known to contain or capable of containing said microorganism; (b) mixing the blood sample with a differential lysis buffer comprising a buffer substance, a non-ionic surfactant, and a salt, said mixing resulting in a lysate comprising lysed blood cells and unlysed microorganisms, wherein the blood sample mixed with the differential lysis buffer has a pH of about 7.0-8.0, and the buffer substance has a useful pH buffering range of about 8.6-11.4; (c) concentrating the microorganisms from the lysate, wherein the microorganisms are concentrated in the range of 25-100 fold relative to the starting volume of the provided blood sample; (d) identifying the microorganisms present in the blood sample, wherein the identification is accomplished in 4 hours or less, 3 hours or less, 2 hours or less, or preferably, 1 hour or less; and A method comprising:

30. 30. The method of claim 29, wherein the identification comprises one or more of a molecular test, a phenotypic test, a proteomic test, an optical test, or a culture-based test.

31. 30. The method of claim 29, wherein the identification comprises isolating from the microorganism one or more nucleic acids characteristic of the microorganism, and analyzing the one or more nucleic acids to identify the microorganism present in the blood sample.

32. 30. The method of claim 29, wherein the non-ionic surfactant is a polyoxyethylene (POE) ether, preferably one or more of Arlasolve 200 (aka poly(oxy-1,2-ethanediyl)), Brij O10, and nonaethylene glycol monododecyl ether (aka Brij 35).

33. 30. The method of claim 29, wherein the non-ionic detergent is selected from the group consisting of Triton X-114, NP-40, Arlasolve 200, Brij O10 (also known as Brij 96 / 97), octyl β-D-glucopyranoside, saponin, nonaethylene glycol monododecyl ether (also known as Brij 35), and combinations thereof.

34. 30. The method of claim 29, wherein the buffering substance is selected from the group consisting of CABS, CAPS, CAPSO, CHES, and combinations thereof.

35. 35. The method of claim 34, wherein the buffering substance is CAPS, and CAPS has a pH buffering range of about 9.7 to 11.1 and a pKa of about 10.4 at 25°C.

36. 30. The method of claim 29, wherein the salt is sodium chloride.

37. 30. The method of claim 29, which does not include a blood culture step prior to said enrichment and / or a DNase step to digest genomic DNA in the lysate.

38. 30. The method of claim 29, wherein steps (a)-(c) are completed within a time range of about 10 to 20 minutes.

39. 32. The method of claim 31, wherein the isolating and analyzing steps can be completed within a time range of about 5 to 75 minutes.

40. The method according to claim 29, wherein the time for obtaining the lysate is within the range of about 2 to 10 minutes, preferably about 5 minutes.

41. 30. The method of claim 29, wherein obtaining the lysate does not include any further steps other than said combining.

42. A blood sample known to contain or capable of containing a microorganism; and a differential lysis buffer that is combined with the blood sample, the differential lysis buffer comprising an aqueous medium, a buffer substance, a non-ionic surfactant, and a salt; wherein the composition has a useful pH buffering range of about 8.6 to 11.4, and has a pH of about 7.0 to 8.0, with said buffering substance having a pKa in the range of about 9.5 to about 10.7 at 25° C.

43. 43. The composition of claim 42, wherein the non-ionic surfactant is a polyoxyethylene (POE) ether, preferably one or more of Arlasolve 200 (aka poly(oxy-1,2-ethanediyl)), Brij O10, and nonaethylene glycol monododecyl ether (aka Brij 35).

44. 43. The composition of claim 42, wherein the non-ionic surfactant is selected from the group consisting of Triton X-114, NP-40, Arlasolve 200, BrijO10 (also known as Brij 96 / 97), octyl β-D-glucopyranoside, saponin, nonaethylene glycol monododecyl ether (C12E9, polidocenol), and combinations thereof.

45. 43. The composition of claim 42, wherein the buffering agent is selected from the group consisting of CABS, CAPS, CAPSO, CHES, and combinations thereof.

46. 46. ​​The composition of claim 45, wherein said buffering substance is CAPS, having said pH buffering range of about 9.7 to 11.1 and said pKa at 25°C of about 10.

4.

47. 43. The composition of claim 42, wherein the buffer substance is substantially positively charged at the pH of about 7.0 to 8.

0.

48. 43. The composition of claim 42, which is free of DNase.

49. said blood sample known to contain or capable of containing microorganisms; said differential lysis buffer comprising a buffer substance; and The nonionic surfactant 43. The composition of claim 42, consisting essentially of, wherein the composition has said pH of about 7.0 to 8.0, with said buffering substance being CAPS, which has said useful pH buffering range of about 9.7 to 11.1 and said pKa at 25° C. of about 10.4.

Images