Methods and devices for rapid isolation and detection of microbial cells

Abstract

A method for the rapid detection of microbial cells is provided. In some embodiments, microbial cells are obtained from a sample, seeded on a solid growth medium, incubated, and monitored for the detection of microcolonies. Microbial cells are harvested from the microcolonies to provide microbial cells for rapid nucleic acid amplification and / or sequencing. Microbial cells can be obtained from a sample containing whole blood by lysing host blood cells with a hemolytic reagent and separating the microbial cells to obtain a suspension. A method is provided for separating microbial cells from a whole blood sample, wherein the whole blood sample is mixed with a hemolytic reagent containing saponin and sodium polyanethole sulfonate to form a first mixture, and then mixed with an alkaline buffer to form a second mixture. A separation process is then used to separate the microbial cells from the second mixture.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 462,857, filed April 28, 2023, titled "METHODS FOR RAPIDLY DETECTING ANTIMICROBIAL RESISTANCE FROM HARVESTED MICROCOLONIES," which is incorporated herein by reference in its entirety, and also claims priority to U.S. Provisional Patent Application No. 63 / 472,222, filed June 9, 2023, titled "METHODS AND DEVICES FOR THE RAPID SEPARATION AND DETECTION OF MICROBIAL CELLS," which is incorporated herein by reference in its entirety, and also claims priority to U.S. Provisional Patent Application No. 63 / 472,222, filed September 11, 2023, titled "METHODS AND DEVICES FOR THE RAPID SEPARATION AND DETECTION OF MICROBIAL CELLS We claim priority to U.S. Provisional Patent Application No. 63 / 537,732, titled "CELLS".

[0002] This disclosure relates to the growth, detection, and characterization of microbial cells. [Background technology]

[0003] Identifying the organisms causing microbial infections and measuring their antimicrobial susceptibility profiles are primary goals of diagnostic process planning in clinical microbiology laboratories. Common practice currently involves collecting patient blood into culture bottles containing antibiotic absorbents, incubating the bottles in an environment that promotes the growth of microbial cell contents, performing Gram staining to classify bacterial cells in terms of cell wall characteristics and morphology, subculturing cells in solid growth media such as agar plates to obtain pure microbial colonies, partially or completely identifying microbial cells, suspending colony contents in culture medium to bring cell concentrations within a desired range, incubating aliquots of the cell suspension in appropriate media with various selected doses of antimicrobial agents, and measuring the minimum inhibitory concentration (MIC) from the cell aliquot growth profile. The main drawbacks of this diagnostic process planning are the time it takes to obtain results (of several days) and the possibility of preferential growth in multimicrobial samples. [Overview of the project] [Means for solving the problem]

[0004] A method for the rapid detection of microbial cells is provided. In some embodiments, microbial cells are obtained from a sample, seeded on a solid growth medium, incubated, and monitored for the detection of microcolonies. Microbial cells from the microcolonies can be harvested to provide microbial cells for subsequent analysis, such as rapid nucleic acid growth and / or sequencing. Microbial cells can be obtained from a sample containing whole blood by lysing host blood cells with a hemolytic reagent and separating the microbial cells to obtain a suspension. A method is provided for separating microbial cells from a whole blood sample, wherein the whole blood sample is mixed with a hemolytic reagent containing saponin and sodium polyanetholesulfonate to form a first mixture, and then mixed with an alkaline buffer to form a second mixture. A separation process is then used to separate the microbial cells from the second mixture.

[0005] Therefore, in the first embodiment, a method for performing nucleic acid amplification using nucleic acids derived from microcolonies of microbial cells, To detect the presence of microcolonies in solid growth medium, The method involves harvesting microbial cells from microcolonies while the diameter of the microcolonies remains less than 100 microns, thereby obtaining the harvested microbial cells. The process involves lysing the recovered microbial cells to obtain a lysate, The method involves performing nucleic acid amplification in the absence of an intervening proliferation step to amplify at least one nucleic acid present in the lysate, thereby obtaining an amplification product. A method including this is provided.

[0006] In some exemplary implementations, the method further includes detecting the presence of amplification products.

[0007] In some exemplary implementations of the method, at least one nucleic acid amplified by nucleic acid amplification contains a drug resistance gene.

[0008] In some exemplary implementations of the method, nucleic acid amplification is configured to amplify multiple nucleic acids.

[0009] In some exemplary implementations of the method, at least one of multiple nucleic acids contains a drug resistance gene.

[0010] In some exemplary implementations of the method, multiple nucleic acids contain a set of drug resistance genes.

[0011] In some exemplary implementations of the method, at least one of the nucleic acids contains a drug resistance gene, and at least one of the nucleic acids is uniquely associated with a microbial species.

[0012] In some exemplary implementations of the method, at least one of the nucleic acids contains a drug resistance gene, and at least one of the nucleic acids is uniquely associated with a microbial genus.

[0013] In some exemplary implementations of the method, at least one of the nucleic acids contains a drug resistance gene, and at least one of the nucleic acids is uniquely associated with a microbial strain.

[0014] In some exemplary implementations of the method, at least two of the nucleic acids are associated with drug resistance genes, and at least two of the nucleic acids are associated with specific microbial genus, strain, or species.

[0015] In some exemplary implementations of the method, at least two nucleic acids are amplified in a multiple amplification reaction.

[0016] In some exemplary implementations of the method, nucleic acid amplification is performed without performing nucleic acid extraction from the lysate.

[0017] In some exemplary implementations of the method, the microbial cells are Gram-positive microbial cells, and the lysis of the microbial cells is carried out by thermal lysis.

[0018] In some exemplary implementations, the method further includes performing sequencing on the amplified product. The nucleic acid amplification step may include whole-genome amplification.

[0019] In some exemplary implementations, the method detects the presence of microcolonies before it detects them. Obtaining a sample, This involves seeding microbial cells from a sample onto a solid growth medium without culturing the sample, Incubate the solid growth medium and monitor the solid growth medium for the presence of one or more microcolonies. It also includes.

[0020] Seeding microbial cells from a sample into a solid growth medium may involve direct contact between the sample and the solid growth medium.

[0021] In some exemplary implementations of the method, the sample is a urine sample.

[0022] In some exemplary implementations, the method involves, before seeding microbial cells from a sample onto a solid growth medium, Microbial cells are isolated from the sample, and the microbial cells are resuspended to obtain a microbial suspension. The method further includes seeding microbial cells from a sample, which involves contacting the microbial suspension with a solid growth medium.

[0023] In some exemplary implementations of the method, the sample contains whole blood, and microbial cells are isolated from the sample. The sample is brought into contact with an alkaline blood lysis reagent containing saponin and sodium polyanethole sulfonate to obtain a mixture. Microbial cells are separated from the mixture, and the microbial cells are resuspended to obtain a microbial suspension. Includes.

[0024] In some exemplary implementations of the method, microbial cells are seeded onto a solid growth medium such that the area ratio of residual sample debris is 10% to 50%.

[0025] In some exemplary implementations of the method, microbial cells are seeded onto a solid growth medium such that the area ratio of residual sample debris is 10% to 40%.

[0026] In some exemplary implementations of the method, microbial cells are seeded onto a solid growth medium such that the area ratio of residual sample debris is 10% to 30%.

[0027] In some exemplary implementations of the method, microbial cells are seeded in a solid growth medium such that the average lateral dimension of the residual sample debris is 1 to 10 micrometers.

[0028] In some exemplary implementations of the method, the first microcolony is detected in the solid growth medium, and the method is as follows: After harvesting the first microcolony, continue monitoring the second microcolony until it grows to a size suitable for performing the downstream assay. To recover the second microcolony, The downstream assay will be performed using microbial cells recovered from the second microcolony. It also includes.

[0029] In some exemplary implementations of the method, downstream assays are selected from matrix-assisted laser desorption / ionization and antimicrobial susceptibility testing.

[0030] In another embodiment, a method for performing nucleic acid amplification using nucleic acids derived from microcolonies of microbial cells, To detect the presence of microcolonies in solid growth medium, To monitor microcolonies and identify when they have grown into colonies containing sufficient biomass to facilitate sequencing, Recovering microbial cells from colonies, The process involves lysing the recovered microbial cells to obtain a lysate, In the absence of the intervening proliferation step and the intervening amplification step, a library for sequencing is prepared using nucleic acids from the lysate. A method including this is provided.

[0031] In some exemplary implementations, the method further includes performing sequencing based on a prepared library.

[0032] In another embodiment, a method for performing Gram staining on microbial cells recovered from a microcolony, To detect the presence of microcolonies in solid growth medium, The method involves harvesting microbial cells from microcolonies while the diameter of the microcolonies remains less than 100 microns, thereby obtaining the harvested microbial cells. Gram staining is performed on the recovered microbial cells in the absence of the intervening growth step. A method including this is provided.

[0033] In another embodiment, a method for performing nucleic acid amplification using nucleic acids derived from microcolonies of microbial cells, To detect the presence of microcolonies in solid growth medium, The process involves recovering material from microcolonies while their diameter remains less than 100 microns, and In the absence of an intervening proliferation step, nucleic acid amplification is performed using nucleic acids present with the recovered material. A method including this is provided.

[0034] In another embodiment, a method for detecting an infectious disease, Obtaining a sample, This involves seeding microbial cells from a sample onto a solid growth medium without culturing the sample, Incubating the solid growth medium and monitoring the solid growth medium for the presence of one or more microcolonies, To detect multiple microcolonies in solid growth medium, Using the number of microcolonies, we can identify indicators associated with the severity of infectious diseases. A method including this is provided.

[0035] In another aspect, a method for monitoring infectious diseases, a) Obtaining a sample, b) Seeding microbial cells from a sample into a solid growth medium without culturing the sample, c) Incubate the solid growth medium and monitor the solid growth medium for the presence of one or more microcolonies, d) Detecting multiple microcolonies in solid growth medium, e) After a time delay, repeat steps a) to d), f) Repeat step e) at least once, g) Using the time dependence of the number of detected microcolonies to monitor the infection and A method including this is provided.

[0036] In another embodiment, a method for processing a whole blood sample suspected of containing microbial cells, The method involves collecting a whole blood sample and mixing the whole blood sample with a hemolytic reagent to obtain a first mixture, wherein the hemolytic reagent contains saponin and sodium polyanetholesulfonate. The first mixture is brought into contact with an alkaline buffer solution, and a second mixture having a viscosity less than that of the first mixture is obtained. The microbial cells are separated from the second mixture to obtain a suspension containing the microbial cells. A method including this is provided.

[0037] In some exemplary implementations of the method, the first mixture is brought into contact with the second mixture after a time delay of 15 minutes to 24 hours, allowing for partial digestion of the blood debris in the first mixture before contact with the alkaline buffer.

[0038] In some exemplary implementations of the method, the first mixture is brought into contact with the second mixture after a time delay of 30 minutes to 24 hours, allowing for partial digestion of the blood debris in the first mixture before contact with the alkaline buffer.

[0039] In some exemplary implementations of the method, the first mixture is brought into contact with the second mixture after a time delay of 40 minutes to 24 hours, allowing for partial digestion of the blood debris in the first mixture before contact with the alkaline buffer.

[0040] In some exemplary implementations of the method, the first mixture is brought into contact with the second mixture after a time delay of 15 minutes to 1 hour, allowing for partial digestion of the blood debris in the first mixture before contact with the alkaline buffer.

[0041] In some exemplary implementations of the method, the first mixture is brought into contact with the second mixture after a time delay of 30 minutes to 1 hour, allowing for partial digestion of the blood debris in the first mixture before contact with the alkaline buffer.

[0042] In some exemplary implementations of the method, the first mixture is brought into contact with the second mixture after a time delay of 40 minutes to 1 hour, allowing for partial digestion of the blood debris in the first mixture before contact with the alkaline buffer.

[0043] In some exemplary implementations of the method, the volume of the whole blood sample is 5 ml to 10 ml.

[0044] In some exemplary implementations, the method further includes lysing microbial cells in a suspension to obtain a lysate, and carrying out nucleic acid amplification to amplify at least one nucleic acid present in the lysate, thereby obtaining an amplification product. The method may further include detecting the presence of the amplification product.

[0045] In some exemplary implementations of the method, at least one nucleic acid amplified by nucleic acid amplification contains a drug resistance gene.

[0046] In some exemplary implementations of the method, nucleic acid amplification is configured to amplify multiple nucleic acids. At least one of the multiple nucleic acids may contain a drug resistance gene. The multiple nucleic acids may contain a set of drug resistance genes. At least one of the multiple nucleic acids may contain a drug resistance gene, and at least one of the multiple nucleic acids is intrinsically associated with a microbial species. At least one of the multiple nucleic acids may contain a drug resistance gene, and at least one of the multiple nucleic acids is intrinsically associated with a microbial genus. At least one of the multiple nucleic acids may contain a drug resistance gene, and at least one of the multiple nucleic acids is intrinsically associated with a microbial strain. At least two of the multiple nucleic acids may each be associated with a drug resistance gene, and at least two of the multiple nucleic acids may each be associated with an intrinsic microbial genus, strain, or species. At least two of the nucleic acids may be amplified in a multiple amplification reaction.

[0047] In some exemplary implementations of the method, nucleic acid amplification is performed without performing nucleic acid extraction from the lysate.

[0048] In some exemplary implementations, the method further includes performing sequencing on the amplification product.

[0049] In some exemplary implementations, the method further includes seeding a suspension of microbial cells onto a solid growth medium and monitoring the solid growth medium for the presence of microcolonies. The method may further include detecting the presence of microcolonies.

[0050] In some exemplary implementations of the method, the composition of the hemolytic reagent and the time delay between forming the first mixture and contacting the first mixture with an alkaline buffer may be selected so that, after seeding the microbial cell suspension onto the solid growth medium, residual blood debris from the suspension partially covers the seeding surface of the solid growth medium with a spatial area ratio of less than 50 percent.

[0051] In some exemplary implementations of the method, the composition of the hemolytic reagent and the time delay between forming the first mixture and contacting the first mixture with an alkaline buffer are selected so that, after seeding the microbial cell suspension onto the solid growth medium, residual blood debris from the suspension partially covers the seeding surface of the solid growth medium with a spatial area ratio of less than 40 percent.

[0052] In some exemplary implementations of the method, the composition of the hemolytic reagent and the time delay between forming the first mixture and contacting the first mixture with an alkaline buffer are selected so that, after seeding the microbial cell suspension onto the solid growth medium, residual blood debris from the suspension partially covers the seeding surface of the solid growth medium with a spatial area ratio of less than 30 percent.

[0053] In some exemplary implementations of the method, the composition of the hemolytic reagent and the time delay between forming the first mixture and contacting the first mixture with an alkaline buffer are selected so that, after seeding the microbial cell suspension onto the solid growth medium, residual blood debris from the suspension partially covers the seeding surface of the solid growth medium with a spatial area ratio of less than 20 percent.

[0054] In some exemplary implementations of the method, the composition of the hemolytic reagent and the time delay between forming the first mixture and contacting the first mixture with an alkaline buffer are selected such that the average size of residual blood debris particles present in the suspension is less than 10 micrometers.

[0055] In some exemplary implementations of the method, whole blood samples are collected in a vacuum container containing hemolytic reagents.

[0056] In some exemplary implementations of the method, microbial cells are separated from the second mixture within one hour after contacting the first mixture with an alkaline buffer.

[0057] In some exemplary implementations of the method, the hemolytic reagent is provided such that, after mixing the hemolytic reagent with a whole blood sample, the first mixture contains saponin at a concentration of 0.75–60 mg / ml and sodium polyanethole sulfonate at a concentration of 0.35–50 mg / ml.

[0058] In some exemplary implementations of the method, the pH of the hemolytic reagent is 3.5–8 before contact with the whole blood sample.

[0059] In some exemplary implementations of the method, the concentration of calcium ions in the hemolytic reagent relative to the dry weight of the saponin is less than 0.5% w / w.

[0060] In some exemplary implementations of the method, the concentration of calcium ions in the hemolytic reagent relative to the dry weight of the saponin is 0.1% w / w to 0.5% w / w.

[0061] In some exemplary implementations of the method, the pH of the alkaline buffer is 8-9.

[0062] In some exemplary implementations of the method, the hemolytic reagent further comprises cyclodextrin having a concentration of 0.1 mM to 20 mM.

[0063] In some exemplary implementations of the method, the hemolytic reagent further comprises cyclodextrin having a concentration of 1–5 mM per 3% w / w saponin.

[0064] In some exemplary implementations of the method, a whole blood sample is collected at the collection site and mixed with a hemolytic reagent, and the first mixture is received at a processing site away from the collection site before being brought into contact with an alkaline buffer.

[0065] In some exemplary implementations, the method further includes transporting a first mixture from a collection point to a processing point.

[0066] In some exemplary implementations of the method, an alkaline buffer is mixed with a first mixture in a fluid cartridge, and automated equipment is used to process the fluid cartridge and carry out the automated separation of microbial cells.

[0067] In some exemplary implementations of the method, the second mixture is formed by contacting a portion of the first mixture with an alkaline buffer.

[0068] In another embodiment, a kit for processing a whole blood sample suspected of containing microbial cells, A container containing a hemolytic reagent, the hemolytic reagent comprising saponin and sodium polyanethole sulfonate, the container having an internal pressure low enough to facilitate the collection of 5 mL to 10 mL of whole blood sample from a subject, thereby forming a first mixture when the whole blood sample is drawn into the container under pressure, and the container and A second container containing an alkaline buffer, wherein the alkaline buffer is configured such that, after mixing the first mixture with the alkaline buffer to form the second mixture, the viscosity of the second mixture is less than the viscosity of the first mixture. A kit including this will be provided.

[0069] In some exemplary implementations of the kit, the concentration of calcium ions in the hemolytic reagent is less than 0.5% w / w.

[0070] In some exemplary implementations of the kit, the concentration of calcium ions in the blood lysis reagent is 0.1% w / w to 0.5% w / w.

[0071] In some exemplary implementations of the kit, the hemolytic reagent further includes cyclodextrin at concentrations ranging from 0.1 mM to 20 mM.

[0072] In another embodiment, a blood lysis collection container is provided, comprising a blood lysis reagent, wherein the blood lysis reagent comprises a saponin and sodium polyanethole sulfonate, and the container has sufficient pressure to facilitate the collection of 5 ml to 10 ml of whole blood sample from a subject, thereby forming a first mixture when the whole blood sample is drawn into the container, and the concentration of calcium ions in the blood lysis reagent is 0.1% to 0.5% w / w.

[0073] In another embodiment, a method for recovering microcolonies from a solid growth medium, Between the solid growth medium and the conductive elongated member, a potential difference is applied such that the conductive elongated member has positive polarity with respect to the solid growth medium. The distal end region of the conductive elongated member is brought close enough to the microcolony to facilitate the collection of microbial cells from the microcolony via an electric field to the distal end region. A method including this is provided.

[0074] In some exemplary implementations of the method, the distal end region includes an electrically insulating layer that prevents direct electrical contact between the conductive portion of the conductive elongated member and the collected microbial cells.

[0075] In some exemplary implementations of the method, the potential difference is between 3V and 50V.

[0076] In another embodiment, a method for recovering microcolonies from a solid growth medium, To provide a fluid transfer device including a fluid transfer tube, wherein the fluid transfer tube has a distal end including an aperture suitable for fluid suction and distribution, Controlling the fluid transfer device to aspirate the recovered buffer solution, The fluid transfer tube is positioned such that its distal end contacts the solid growth medium with the aperture around the microcolony, thereby surrounding the microcolony and forming a seal between the fluid transfer tube and the solid growth medium. The process involves distributing at least a portion of the recovered buffer, controlling a fluid transfer device to distribute the recovered buffer so that at least a portion of it enters a solid growth medium, thereby releasing microbial cells from microcolonies and forming a suspension containing the released microbial cells. Controlling the fluid transfer device to aspirate at least a portion of the suspension and A method including this is provided.

[0077] In some exemplary implementations of the method, the distal end of the fluid transfer device is located below the surface of the solid growth medium during the distribution of the recovered buffer and the aspiration of the suspension. The distal end of the fluid transfer device may extend to a depth of 0.1–0.6 mm below the surface of the solid growth medium. The distal end of the fluid transfer device may extend to a depth of 0.1–0.4 mm below the surface of the solid growth medium. The distal end of the fluid transfer device may extend to a depth of 0.1–0.6 mm below the surface of the solid growth medium.

[0078] A further understanding of the functional and advantageous aspects of this disclosure can be achieved by referring to the following detailed description and drawings.

[0079] Embodiments will be described with reference to the attached drawings. In the drawings, the same reference number may represent the same or functionally similar component. The drawing in which a component first appears is generally indicated by the leftmost digit of the corresponding reference number. [Brief explanation of the drawing]

[0080] [Figure 1A] This flowchart illustrates an exemplary method for rapidly detecting drug resistance markers, based on recovering microbial cells from microcolonies directly grown from a sample and performing nucleic acid amplification using nucleic acids released from the recovered microbial cells. [Figure 1B] This flowchart illustrates an exemplary method for rapidly performing nucleic acid amplification assays based on microbial cells recovered from microcolonies directly grown from a sample. [Figure 2] This flowchart illustrates an exemplary method for rapidly performing sequencing based on microbial cells recovered from microcolonies directly grown from a sample. [Figure 3] This represents the average time to positive results and the diameter of microcolonies for several microbial species. [Figure 4-1] This represents the number of bacterial cells harvested from microcolonies incubated 4 hours after expansion of the cell suspension. Each data point is averaged across five microcolonies. [Figure 4-2] Figure 4B schematically illustrates an exemplary method for performing microcolony retrieval using electrical attraction and repulsion. Figure 4C shows the number of bacterial cells retrieved from incubated microcolonies using aspiration and electrorecovery methods 4 hours after cell suspension expansion. Each data point is averaged across five microcolonies. [Figure 5]This image shows Gram-stained E. coli ATCC 35218 microcolonies (4-hour incubation) recovered using affinity surfaces with various adhesive materials. [Figure 6] This image shows Gram-stained Staphylococcus aureus ATCC 25923 microcolonies (4-hour incubation) recovered using affinity surfaces with various adhesive materials. [Figure 7] This represents the time to a positive result in the RT-LAMP assay performed on cell suspensions recovered from Staphylococcus aureus and Escherichia coli microcolonies. [Figure 8] This represents the time to a positive result in the RT-LAMP assay performed on cell suspensions collected from Staphylococcus aureus and Escherichia coli microcolonies grown for 4 hours after expansion. [Figure 9A] This represents the time to a positive result in the RT-LAMP assay performed on cell suspensions collected from microcolonies grown for 4 hours after expansion. [Figure 9B] This represents the time to positivity in the RT-LAMP assay performed on two aliquots of cell suspension collected from microcolonies grown for 4 hours after expansion. One aliquot was subjected to thermal lysis, while the other was tested without lysis. [Figure 10] Figure 10A shows the CT values ​​in PCR assays performed on cell suspensions recovered from Staphylococcus aureus ATCC 29223 colonies grown for 4 or 6 hours after expansion. Figure 10B compares the CT values ​​in PCR assays performed on two aliquots of cell suspensions recovered from Escherichia coli and Staphylococcus aureus microcolonies grown for 4 hours after expansion. [Figure 11-1]Figure 11A shows sections of blood agar plates imaged using an upright episcopic bright-field (BF) metallurgical microscope with a 5x infinite flattening objective lens. Microbial cell suspensions obtained from whole blood, which were selectively dissolved with a hemolytic reagent consisting of saponin and sodium polyanetholesulfonate (SPS), and then subjected to two centrifugation washing cycles, were distributed onto plates, air-dried, and microscopic images were obtained. Regions where the sample grew are indicated by 312. Figure 11B shows sections of blood agar plates imaged using an upright episcopic bright-field (BF) metallurgical microscope with a 5x infinite flattening objective lens. Microbial cell suspensions obtained from whole blood, which were selectively dissolved with a hemolytic reagent containing saponin, SPS, Triton-X100, and carbonate-bicarbonate buffer, and then subjected to two washing cycles, were distributed onto plates, air-dried, and microscopic images were taken. [Figure 11-2] This figure shows the size distribution of blood debris obtained using the hemolytic reagents used when processing samples according to the method described in Figure 3B. A 1 μL microbial cell suspension obtained from whole blood was selectively dissolved with a hemolytic reagent containing saponin, SPS, Triton-X100, and carbonate-bicarbonate buffer, and then subjected to 2 or 4 washing cycles. The suspension was then plated, air-dried, and microscopic images were taken using a 10x infinite flattening objective lens. The particle size distribution was analyzed from the images, and histograms of the particle size distribution were plotted for 2 washing cycles (left) and 4 washing cycles (right). [Figure 12] The images show sections of microculture regions (MCRs) formed on agar plates after distributing 1 μL of microbial cell suspension obtained by centrifugation of whole blood samples spiked with Proteus mirabilis (PM), imaged with a bright-field (BF) metallurgical microscope equipped with a 5x infinite planarization objective lens at 0, 2, 3, and 4 hours of incubation. Arrows indicate blood lysis debris and some PM microcolonies identifiable by sight. [Figure 13]Time-lapse image analysis illustrates the exemplary steps of differentiating microbial colonies from blood-lysic debris using MCR (Microbial Regeneration) as shown in Figure 6. Imaging data acquired at various time points (0, 2, 3, and 4 hours after seeding) were spatially aligned (registered) with the 0-hour image, and then the 0-hour image was subtracted. Intensity traits present in the 0-hour image were classified as background (blood-lysic debris), while intensity traits appearing in the subtracted image were classified as foreground microcolonies. [Figure 14-1] Figure 14A is a table showing the measured growth parameters of seeded ATCC strains of Gram-positive bacteria recovered from blood samples spiked by centrifugation and subsequent seeding on agar. It shows the pre-growth lag time, growth rate, estimated time to positivity, and the average time required for a large number of cells in a microcolony to reach 10⁴ and 10⁵ CFU, relative to seeded cell growth in blood culture bottles and reference growth (liquid culture medium). Figure 14B is a table showing the measured growth parameters of seeded clinical isolates of Gram-positive bacteria recovered from blood samples spiked by centrifugation and subsequent seeding on agar. It shows the pre-growth lag time, growth rate, estimated time to positivity, and the average time required for a large number of cells in a microcolony to reach 10⁴ and 10⁵ CFU, relative to seeded cell growth in blood culture bottles and reference growth (liquid culture medium). Figure 14C is a table showing the measured growth parameters of further seeded clinical isolates of Gram-positive bacteria recovered from blood samples spiked by centrifugation and subsequent seeding on agar. The lag time before proliferation, proliferation rate, estimated time to positivity, and the average time required for a large number of cells in a microcolony to reach 10⁴ and 10⁵ CFU are expressed relative to seeded cell proliferation in blood culture bottles and reference proliferation (liquid culture medium). [Figure 14-2]Figure 14D is a table showing the measured growth parameters of seeded ATCC strains of Gram-negative bacteria recovered from blood samples spiked by centrifugation and subsequent seeding on agar. It shows the pre-growth lag time, growth rate, estimated time to positivity, and the average time required for a large number of cells in a microcolony to reach 10⁴ and 10⁵ CFU, relative to seeded cell growth in blood culture bottles and reference growth (liquid culture medium). Figure 14E is a table showing the measured growth parameters of seeded clinical isolates of Gram-negative bacteria recovered from blood samples spiked by centrifugation and subsequent seeding on agar. It shows the pre-growth lag time, growth rate, estimated time to positivity, and the average time required for a large number of cells in a microcolony to reach 10⁴ and 10⁵ CFU, relative to seeded cell growth in blood culture bottles and reference growth (liquid culture medium). Figure 14F is a table showing the measured growth parameters of additional seeded clinical isolates of Gram-negative bacteria recovered from blood samples spiked by centrifugation and subsequent seeding on agar. The lag time before proliferation, proliferation rate, estimated time to positivity, and the average time required for a large number of cells in a microcolony to reach 10⁴ and 10⁵ CFU are expressed relative to seeded cell proliferation in blood culture bottles and reference proliferation (liquid culture medium). [Figure 14-3] Figure 14G is a table showing the measured growth parameters of seeded ATCC strains of fungi recovered from blood samples spiked by centrifugation and subsequent seeding on agar. The table shows the lag time before growth, growth rate, estimated time to positivity, and the average time required for a large number of cells in a microcolony to reach 10⁴ and 10⁵ CFU, relative to seeded cell growth in blood culture bottles and reference growth (liquid culture medium). Figure 14H is a table showing the measured growth parameters of seeded clinical isolates of fungi recovered from blood samples spiked by centrifugation and subsequent seeding on agar. The table shows the lag time before growth, growth rate, estimated time to positivity, and the average time required for a large number of cells in a microcolony to reach 10⁴ and 10⁵ CFU, relative to seeded cell growth in blood culture bottles and reference growth (liquid culture medium). [Figure 15-1]Figure 15A plots the number of colony-forming units (CFUs) of Staphylococcus epidermidis bacterial cells recovered after centrifugation of microbial cells from spiked whole blood samples and subsequent seeding on agar, following seeding of the final cell suspension and incubation at 37°C for 4 hours. Figure 15B plots the number of CFUs of Staphylococcus epidermidis bacterial cells recovered after centrifugation of microbial cells from spiked whole blood samples and subsequent seeding on agar, following seeding of the final cell suspension and incubation at 37°C for 4 hours. [Figure 15-2] Figure 15C plots the number of CFUs of Pseudomonas aeruginosa bacterial cells recovered after centrifugation of microbial cells from spiked whole blood samples and subsequent seeding on agar, at different time points, after seeding the final cell suspension and incubation at 37°C for 4 hours. Figure 15D plots the number of CFUs of Escherichia coli bacterial cells recovered after centrifugation of microbial cells from spiked whole blood samples and subsequent seeding on agar, at different time points, after seeding the final cell suspension and incubation at 37°C for 6 hours. [Figure 16] This shows a positive optical microscope measurement of spiked blood samples against Candida albicans cells (visible inner oval) after isolation from whole blood samples and incubation for 4 hours. [Figure 17-1] Figure 17A shows a plot of the average diameter and microcolony cell content of E. coli. The plot is fitted with an exponential trend line to allow estimation of the average microcolony diameter at, for example, 10³ and 10⁵ cell content levels. [Figure 17-2] Figure 17B shows the average microcolony diameters at 10³ and 10⁵ cell contents for various pathogenic Gram-positive bacteria present in bloodstream infections. Figure 17C shows the average microcolony diameters at 10³ and 10⁵ cell contents for various pathogenic Gram-negative bacteria present in bloodstream infections. [Figure 18]This flowchart illustrates a whole blood sample preparation method in which a hemolytic reagent containing saponin and sodium polyanethole sulfonate (SPS) is mixed with an alkaline buffer to form an alkaline hemolytic reagent, which is then brought into contact with a whole blood sample, and subsequently, microbial cells are separated from the mixture using a separation process. [Figure 19] This flowchart illustrates an improved whole blood sample preparation method, in which a whole blood sample is contacted and mixed with a hemolytic reagent containing saponin and SPS to form a first mixture, the first mixture is then contacted with an alkaline buffer to obtain a second mixture, and then microbial cells are separated from the second mixture using a separation process. [Figure 20] The graphs show the time dependence of viscosity (top) and microscopic morphology (bottom) of a mixture obtained by mixing a whole blood sample with a hemolytic reagent containing saponin and SPS, as well as the effect of contact with alkaline buffer after a time delay. [Figure 21] This table shows the results from experimental studies on the volume of whole blood, the volume of hemolytic reagent, the delay in mixing saponin and SPS-containing hemolytic reagent with alkaline buffer, and the dependence of the pH of the final mixture of hemolytic reagent, whole blood sample, and alkaline buffer on the separation of solid growth medium and the particle debris size and surface coverage after seeding. [Figure 22] Following the method shown in Figure 19, microscopic images of cell suspensions seeded on agar plates after processing whole blood samples are shown, along with the results for different concentrations of calcium in the hemolytic reagent. [Figure 23] The table in Figure 19 shows the dependence of microcolony detection on time in solid growth medium after processing whole blood samples according to the method shown, illustrating the effect of calcium concentration in the hemolytic reagent on microcolony detection and its time dependence. [Figure 24-1] This table shows the compositions of exemplary hemolytic reagents used in the experimental verification of cell recovery and molecular amplification. [Figure 24-2]Figure 24B shows the treated whole blood sample after the third wash and before removing the top 1.9 mL of supernatant. In the case of hemolytic reagent without K2EDTA, a reddish pellet is observed at the bottom of the container. Figure 24C shows a microscopic image of the cell suspension seeded on an agar plate after processing the whole blood sample according to the method shown in Figure 19, and shows the results for two different concentrations of K2EDTA in the hemolytic reagent. [Figure 24-3] From the perspective of providing a clear eluate, this study demonstrates the effect of the presence or absence of K2EDTA in the hemolytic reagent on its performance in the collection tube. [Figure 25] This shows the recovery of various microbial cells from a 2:6 mixture of hemolytic reagent and whole blood collected in an SPS blood tube. [Figure 26] This shows RT-LAMP inhibition in the amplification of Klebsiella pneumoniae by blood matrix obtained by treating 8 mL of unspiked blood with several exemplary hemolytic reagent formulations. "BLR-1 Ctrl" and "BLR-2 Ctrl" represent the amplification of Klebsiella pneumoniae lysates in clear phosphate buffer. Each bar represents 20 different blood samples (donors) for a total of 20 samples, according to mean ("x") TTP, median, interquartile region, maximum, and minimum values. [Figure 27] This represents the difference in time to positivity (TTP) in the RT-LAMP assay between a cell suspension obtained by processing a spiked blood sample and a positive control. Each TTP represents the average of 4-6 RT-LAMP reactions. [Figure 28-1] Figure 28A schematically illustrates an exemplary aspiration method for collecting microcolonies. Figure 28B shows microscopic images of microcolonies before and after collection by the aspiration method in Figure 28A, without disturbing adjacent microcolonies. [Figure 28-2] This represents the sensitivity of an exemplary aspiration retrieval method at the tip position relative to the gel surface. [Figure 28-3] This shows the reproducibility of the aspiration method in Figure 18A for microcolony collection. [Modes for carrying out the invention]

[0081] Various embodiments and aspects of this disclosure will be described with reference to the details discussed below. The following description and drawings of this disclosure are illustrative and should not be construed as limiting the disclosure. Many specific details are provided to allow for a full understanding of the various embodiments of this disclosure. However, in certain examples, well-known or prior details are not described in order to provide a precise discussion of the embodiments of this disclosure.

[0082] As used herein, the terms “includes” and “contains” should be interpreted as inclusive and open-ended, and not exclusive. Specifically, as used herein and in the claims, the terms “includes” and “contains,” and variations thereof, mean that the specified features, steps, or components are included. These terms should not be interpreted as excluding the presence of other features, steps, or components.

[0083] As used herein, the term “exemplary” means “serving as an example, illustration, or diagram,” and should not be construed as being preferable or advantageous to any other structure disclosed herein.

[0084] As used herein, the terms “about” and “approximately” mean that the variation may exist within the upper and lower limits of a range of values ​​such as properties, parameters, and dimensions. Unless otherwise specified, the terms “about” and “approximately” mean ±25% or less.

[0085] Unless otherwise specified, any specified scope or group should be understood as an abbreviation for each and all elements of a scope or group, and for any sub-scope or sub-group, for each and all possible sub-scopes or sub-groups contained therein and similarly contained therein. Unless otherwise specified, this disclosure expressly invokes this with respect to each and all specific elements and combinations of sub-scopes or sub-groups.

[0086] As used herein, the term “order” when used with a quantity or parameter refers to a range of approximately one-tenth to ten times the quantity or parameter being described.

[0087] definition Unless otherwise specified, all technical and scientific terms used herein are intended to have the same meanings as those commonly understood by those skilled in the art. Unless otherwise specified, such as in the context in which they are used herein, the following terms are intended to have the following meanings:

[0088] As used herein, the term “colony” refers to a multiplicity or population of microorganisms that exist adjacent to one another, on a surface, and are clonal descendants of an ancestral microorganism, resulting from in situ replication.

[0089] As used herein, the term "microcolony" refers to a colony having a diameter of less than 100 μm. For non-circular microcolonies, the diameter is the effective diameter of the microcolony, which can be calculated as the diameter of a circle having the same area as the spatial region associated with the microcolony (for example, it can be calculated as √4·area / π).

[0090] As used herein, the term "microcolony growth phase" refers to the period during which a growing colony is a microcolony.

[0091] As used herein, the term "cell suspension" refers to an aqueous medium containing microbial cells.

[0092] As used herein, the term "separation process" refers to a process suitable for separating and optionally connecting microbial cells. Non-limiting examples of separation processes include centrifugation, filtration, immunomagnetic separation, and microfluidic separation.

[0093] As used herein, the term “sample” means a liquid or suspension containing, potentially containing, or suspected to contain one or more microbial cells. Non-limiting examples of samples include bodily fluids such as urine, lymph, cerebrospinal fluid, blood (e.g., whole blood, blood cultures, and plasma), sputum, mucus, wound drainage, saliva, lavage fluid, joint fluid, abscess fluid, homogenized tissue suspensions (including, but not limited to, homogenized suspensions of feces, muscle tissue, brain tissue, and liver tissue), and any fluid aspirates or tissue extracts of human and / or other mammalian organs. Samples may be processed or unprocessed and may optionally contain one or more reagents or growth media.

[0094] As used herein, the term "blood cells" refers to mammalian cells present in the blood, including but not limited to red blood cells, white blood cells, and platelets.

[0095] As used herein, the term "blood sample" refers to any sample containing one or more blood cells. Non-limiting examples of blood samples include whole blood samples, blood culture samples, pia mater samples, and platelet samples.

[0096] As used herein, the terms “whole blood” or “whole blood sample” refer to mammalian blood, including plasma and blood cells. “Whole blood” or “whole blood sample” may include one or more reagents, such as anticoagulants. For example, as described later in some exemplary embodiments of this disclosure, whole blood may be collected in a sample bottle that may contain one or more reagents, such as anticoagulants including, but not limited to, SPS (sodium polyanetholesulfonate), EDTA (ethylenediaminetetraacetic acid), sodium citrate, and heparin.

[0097] As used herein, the term "selective lysis" refers to a hemolytic reagent or lysis process in which the fraction of microbial cells that remain viable after lysis is greater than the fraction of eukaryotic cells that remain viable after lysis, and eukaryotic cells are associated with the subjects from which the sample was collected.

[0098] As used herein, the term "effective buffer concentration," when referring to a mixture formed by mixing a certain volume of sample with a certain volume of hemolytic reagent (the hemolytic reagent includes a buffer system), refers to the ratio formed by dividing the volume of the hemolytic reagent product and the volume of the hemolytic reagent by the sum of the volumes of the hemolytic reagent and the sample. The effective buffer concentration represents the contribution of the hemolytic reagent to the buffer system in the final mixture (i.e., the dilution factor applied to the buffer concentration of the hemolytic reagent). The effective buffer concentration may differ from the actual buffer concentration in the final mixture due to buffering of components present in the sample.

[0099] As used herein, the terms “microbial cells” and “microorganisms” refer to bacteria (e.g., Gram-positive and Gram-negative bacteria and bacterial spores) and single-celled fungi (such as yeasts and filamentous fungi).

[0100] As used herein, the term “eukaryotic cell” refers to cells derived from blood-containing animals, including eukaryotic organisms other than fungi, such as animals, particularly invertebrates such as crustaceans and vertebrates. As used herein, “vertebrate” includes both cold-blooded animals (fish, reptiles, and amphibians) and warm-blooded animals (birds and mammals).

[0101] As used herein, the term "uncultured sample" refers to any sample suspected to contain viable microbial life, wherein the sample has not been incubated with heat in a controlled thermal environment suitable for promoting microbial growth. An "uncultured sample" may include a growth medium. An example of an uncultured sample is a blood culture bottle containing a blood sample, wherein the blood culture bottle has not been incubated in a thermal incubator.

[0102] As used herein, the term “nucleic acid amplification” refers to a process for amplifying or multiplexing one or more nucleic acid molecules. In some embodiments, one or more nucleic acid molecules are derived from pathogens such as viruses, bacteria, or fungal pathogens. Nucleic acid molecules can be amplified according to several different nucleic acid amplification modalities, including, but not limited to, polymerase chain reaction (PCR), real-time PCR, strand displacement amplification (SDA), transcription-mediated amplification (TMA), quantitative PCR (qPCR), reverse transcription PCR (RT-PCR), loop-mediated isothermal amplification (LAMP), RT-LAMP, strand displacement amplification (SDA), helicase-dependent amplification (HDA), transcription-mediated amplification (TMA), recombinase polymerase amplification (RPA), nucleic acid sequence-based amplification (NASBA), and variations thereof. In some exemplary nucleic acid amplification modalities, nucleic acid amplification may be carried out by or using thermal cycling, while in other exemplary modalities, nucleic acid amplification may be carried out by or using isothermal amplification.

[0103] As used herein, the term “target nucleic acid” refers to a nucleic acid containing the target sequence to be amplified and / or detected. The target nucleic acid may be DNA or RNA, and may be single-stranded or double-stranded. The target nucleic acid may contain other sequences besides the target sequence that cannot be amplified.

[0104] As used herein, the term “target sequence” refers to a specific nucleotide sequence of the target nucleic acid to be amplified and / or detected.

[0105] As used herein, the term "buffer solution" refers to any pH-controlled solution that may serve as a dissolving agent for solid substances (e.g., reagents, samples, or combinations thereof) or as a diluent for diluting liquids (e.g., liquid reagents, liquid samples, or combinations thereof, or solutions of reagents, samples, or combinations thereof).

[0106] As used herein, “polymer” refers to an enzyme that uses an existing nucleic acid molecule (e.g., DNA or RNA) as a template to catalyze the formation of a new nucleic acid molecule (e.g., DNA or RNA) and to create a complementary (or substantially complementary) polynucleotide sequence within the new molecule. In some embodiments, the polymerase is a strand substitution polymerase that catalyzes the substitution of one strand of a DNA double helix before DNA or RNA synthesis.

[0107] As used herein, "primer" refers to a polynucleotide that plays a role in initiating a nucleic acid chain elongation reaction.

[0108] As used herein, “probe” refers to a polynucleotide that can hybridize (e.g., specifically) to a target sequence within a nucleic acid under conditions that enable hybridization, thereby enabling the detection of the target sequence or amplified nucleic acid.

[0109] As used herein, the term "multiplex PCR" refers to a type of PCR in which two or more sets of primers are included in the reaction, enabling amplification of a single target or two or more different targets in a single reaction tube. Multiplex PCR may, for example, be real-time PCR.

[0110] As used herein, the term “symptomatic panel” refers to a multiplex assay that simultaneously detects multiple different pathogens associated with similar and / or overlapping clinical symptoms.

[0111] As used herein, the term "solid growth medium" refers to a growth medium on which microorganisms can colonize. An example of a solid growth medium is a gel, such as an agar-based gel, which is a colloidal system in which a porous network of interconnected particles diffuses nutrients through the medium over the volume of the liquid medium, making them available to microorganisms.

[0112] As used herein, the term “host cell” refers to a cell originating from a different host or subject than symbiotic animal cells (e.g., microbial cells that are part of the host microbiome), infectious cells (e.g., pathogenic microorganisms), or contaminated cells (e.g., accidentally introduced during sample collection or preparation). The term “host” may refer to a patient or medical subject that may be human or a non-human mammal.

[0113] As used herein, the term "saponin" refers to steroid saponins, triterpenoid saponins, and / or combinations thereof.

[0114] introduction As described above, conventional diagnostic workflows for identifying and measuring the microbial organisms responsible for infectious diseases are fraught with challenges and delays. In fact, the time delay before microbial identification and antimicrobial susceptibility results become available is typically several days, resulting in current standard care for treating microbial infections being primarily based on empirical treatments. Consequently, identification and antimicrobial susceptibility results are often communicated too late to have a clinical impact on individual patients.

[0115] To address these shortcomings, the inventors have developed a novel approach to the detection, identification, and antimicrobial susceptibility testing of blood samples. In contrast to conventional workflows that rely on blood cultures, the inventors have developed a novel approach based on the direct growth of microcolonies. In such a microcolony-based method, a blood sample is processed to obtain a concentrated suspension of microbial cells, and this concentrated suspension is distributed into a solid growth medium. The solid growth medium is incubated to grow microbial colonies, which are automatically detected by microscopic imaging during the initial microcolony stage.

[0116] In the international patent specification PCT / CA2019 / 051895, titled "SYSTEMS AND METHODS FOR MICROCOLONY GROWTH AND MICROBIAL CELL CHARACTERIZATION," which is incorporated herein by reference in its entirety, the inventors demonstrated that microcolony detection can be achieved within a few hours for a diverse range of bacterial and fungal species. This rapid microcolony-based detection in the presence of infectious diseases represents a significant new development and improvement over conventional blood culture-based approaches for detection.

[0117] The inventors also describe a method for recovering microbial cells from growing microcolonies and using them for identification of microbial cells by MALDI (matrix-assisted laser desorption / ionization) and / or for use in antimicrobial susceptibility testing (AST). Specifically, International Patent PCT / CA2019 / 051895 describes a method for monitoring the size of growing microcolonies by intermittent microscopic imaging, and microbial cells are recovered from microcolonies that exceed a predetermined size threshold associated with a sufficient quantity of microbial cells to perform MALDI and / or AST, by identifying indicators associated with microcolony size. As described in International Patent PCT / CA2019 / 051895, it has been observed that microcolonies grow to contain a sufficiently large number of microbial cells for MALDI and / or AST within approximately 5 hours for a wide range of microbial species. Since MALDI results become available within one hour, and new rapid AST methods (such as the local nucleic acid AST method described by the inventors in international patent PCT / CA2019 / 051895) can deliver MIC results within a few hours, microcolony-based methods may be able to deliver sufficient phenotypic AST results within a single shift.

[0118] The inventors have further developed this idea by including a pre-microcolony retrieval presumptive identification step, which involves processing dark-field images obtained at various points during the microcolony growth phase using a machine learning algorithm to provide an initial measurement of microbial cell class, such as genus and / or species determination. Such a method of presumptive identification is disclosed in international patent application PCT / CA2021 / 050884, entitled "SYSTEMS AND METHODS FOR CLASSIFICATION OF MICROBIAL CELLS GROWN IN MICROCOLONIES," which is incorporated herein by reference in its entirety. Presumptive measurement of microbial cell class can be used, for example, as an initial means of identification and / or for selecting an appropriate subset of antimicrobial agents for antimicrobial susceptibility testing.

[0119] Therefore, the direct microcolony-based method previously disclosed by the inventors in international patent applications PCT / CA2019 / 051895 and PCT / CA2021 / 050884 provides a complete workflow that can provide (i) rapid detection of the presence of infection without several hours of sample collection based on direct observation of microcolonies, (ii) initial presumptive identification based on image-based classification, (iii) rapid complete microbial identification by performing MALDI on microbial cells recovered from microcolonies, and (iv) the same shift phenotypic AST performed on microbial cells recovered from microcolonies (resulting in minimum inhibitory concentration results), enabling susceptibility / intermediate / resistance measurements to be made based on AST and MALDI ID results and establishing clinical breakpoints. This approach therefore appeared to provide a complete solution to the challenges associated with conventional workflows in microbiology.

[0120] However, despite the practicality of direct microcolony-based workflows that utilize MALDI and AST as described above to yield identical-shift phenotypic AST results from recovered microcolonies, we have recently come to recognize that direct microcolony-based methods can offer additional clinical value by facilitating the detection of drug resistance genes before conveying identical-shift complete phenotypic AST results. Indeed, while the detection of the presence of an infection is perhaps the most clinically relevant outcome available—a result that is indeed provided rapidly and clearly by direct microcolony-based methods by directly detecting the growth of microcolonies—the next most relevant and clinically useful information is the measurement of whether or not infectious microbial pathogens exhibit drug resistance.

[0121] While same-shift full phenotypic AST results directly address the presence or absence of drug resistance, the inventors sought an approach that facilitates the clear measurement of drug resistance early in the workflow, immediately after detecting the presence of an infection.

[0122] Molecular detection of drug resistance is conventionally performed using positive blood culture samples containing very large numbers of microbial cells. This approach has been successfully commercialized as multi-severity blood culture identification panels, such as the BioFire® FilmArray® BCID panel, Roche® ePlex®, and Luminex® Veigene® Bloodstream Infection Testing Panels, each containing genomic resistance markers.

[0123] At the opposite end of the spectrum, attempts have been made to use nucleic acid amplification to directly instruct small amounts of molecular pathogens in whole blood samples. For example, T2 Biosystems® has developed the T2Resistance® panel, which directly detects resistance genes in microbial pathogens isolated from whole blood.

[0124] The inventors recognized that both approaches to detecting resistance markers, namely the positive blood culture approach and the direct whole blood approach, have significant drawbacks. The positive blood culture approach benefits from a large number of microbial cells, facilitating clear and unambiguous detection of resistance markers. Furthermore, the positive blood culture approach is cost-effective because positivity is already measured before the assay is performed. On the other hand, the positive blood culture approach is affected by a significant time delay that occurs before the blood culture becomes positive, often resulting in resistance marker results being delayed until day 2. Therefore, while such multi-severity symptom panels are rapid in terms of assay time and assay results, they are clinically delayed due to the delay in blood culture positivity.

[0125] In contrast, the direct whole blood approach to detecting resistance markers is superior in that it yields resistance marker results very rapidly, often within hours of sample collection. Unfortunately, the direct whole blood approach to detecting resistance markers is ultimately affected by significant drawbacks that challenge its adoption. One drawback of direct whole blood detection is the deficiency of microbial cells in blood samples, which often involves low microbial concentrations at the 1 CFU / mL level. This low target places a heavy load on assay techniques, causing sensitivity to approach its limits and creating a risk of false-positive detection. Another challenge with whole blood samples is that host blood cells, which can number eight or nine orders of magnitude more than bacterial genetic material in terms of nucleic acid counts including cell-free DNA, significantly outnumber the target microbial cells.

[0126] These challenges, combined with the low symptomatic rate of positive whole blood samples, result in a positive rate typically ranging from 5% to 10%. While this low symptomatic rate inherently leads to a high negative predictive value, the small sample size effectively amplifies the risk of false positive detection. Since the total symptomatic rate includes both (i) the number of resistance markers among positive samples and (ii) the number of positive samples among all samples, in a direct detection symptomatic panel testing a large number of resistance markers, for example, the total symptomatic rate for detecting a given resistance marker may be less than 1%. Therefore, even a false positive rate of 1% can result in more false positives than true positives, hindering the ability to clinically utilize such tests for early treatment decision-making.

[0127] Rapid detection of drug resistance using microcolony-based methods. Recognizing the significant shortcomings of both positive blood culture and direct whole blood approaches for resistance marker detection, the inventors pursued an improved approach that captures the advantages of both conventional approaches while avoiding their drawbacks. In other words, the inventors pursued a novel approach to resistance marker detection that (i) includes an initial detection step (excluding negative samples) to determine which samples were positive, (ii) provides results within a few hours of receiving the samples, and (iii) performs robust detection of a sufficiently large number of microbial cells to avoid the challenges of false positives.

[0128] The inventors recognized that this objective could be achieved by employing a direct microcolony-based approach to perform nucleic acid detection and / or sequencing of microbial cells recovered from microcolonies. Such microcolony-based nucleic acid detection methods may be able to satisfy the requirements outlined above. As detailed below, a key factor in achieving this was recognizing that microcolonies provide a highly localized and purified source of microbial cells, even in the early stages, that can be efficiently recovered for nucleic acid detection and / or sequencing in the absence of an intervening microbial growth step between recovery and nucleic acid amplification and / or sequencing.

[0129] In fact, the microcolony-based detection method described herein facilitates the direct detection of positive samples in just a few hours based on initial microscopic imaging during microcolony growth, thereby enabling the identification of positive samples before nucleic acid detection. Therefore, the microcolony-based nucleic acid detection method solves the "negative challenges" and enables cost-effective nucleic acid detection using only positive samples.

[0130] Furthermore, unlike liquid culture-based approaches to detection, this microcolony-based method, which does not involve liquid culture-based incubation of the sample where microcolonies grow from microbial cells obtained from the sample, allows for the measurement of quantitative indicators based on the number of microcolonies detected. For example, the number of detected microcolonies can be used to infer the severity of an infection and / or the effectiveness of antimicrobial treatment. Using this quantitative aspect of the method, the severity of an infection or the effectiveness of antimicrobial treatment can also be tracked over time based on detections performed on multiple samples at various time points. This direct aspect of microcolony-based detection may also be beneficial in facilitating the measurement of sample-specific and true polymicrobiality, which is not interfered with or biased by competition, such as masking or distorting the relative concentrations of subpopulations of different types of microbial cells, as can occur in other liquid culture environments.

[0131] Furthermore, as detailed below, this microcolony-based detection allows for the rapid initiation of nucleic acid amplification and / or sequencing immediately after microcolony detection, without requiring further growth (or further cell enrichment). In fact, it will be shown that even very small microcolonies, such as those with a horizontal scale of 20–50 microns, typically contain sufficient microbial cells for efficient and robust nucleic acid detection. Considering that microcolony detection and recovery can be performed within a few hours of sample processing initiation, and the subsequent nucleic acid amplification step can often be performed within an hour (sequencing results are available within 2–3 hours), the microcolony-based resistance marker detection method can provide resistance marker results within a few hours of sample collection, and can easily fit within a single shift.

[0132] Furthermore, the inventors recognized that microcolony-based detection methods offer significant advantages in achieving levels of concentration and isolation that are unattainable using either conventional approaches to positive blood culture detection or direct whole blood detection. In fact, in the case of positive blood culture detection, the sample is a complex suspension of microbial cells, growth medium, blood debris, and other components of the blood culture medium, such as antimicrobial absorbent resins. Each of the aforementioned commercial products, while having the advantage of a high microbial cell load in the initial sample, results in complex microbial cell isolation and nucleic acid extraction steps. In the case of direct whole blood processing, the low prevalence of microbial cells in the blood sample makes the sample preparation steps necessary to isolate microbial cells and extract nucleic acids extremely important. In fact, the inventors found that processing a sufficiently large volume of whole blood to facilitate the detection of a single CFU / mL sample while simultaneously reducing the concentration of nucleic acid interferants, achieving high microbial cell recovery, and sufficient concentrations of both microbial cells and isolated microbial cells can be extremely difficult. Such challenges can be a source of obstacles and deficiencies in commercially available whole blood nucleic acid detection and / or sequencing solutions.

[0133] In stark contrast to these limitations in conventional approaches, this microcolony-based nucleic acid detection method achieves improved isolation and concentration levels through the microcolony modality. Unlike conventional direct whole blood nucleic acid amplification approaches, which are performed entirely in the liquid phase, the intermediate solid growth step in the microcolony-based nucleic acid detection method efficiently results in improved spatial and growth-based isolation and concentration.

[0134] In fact, the microcolony-based nucleic acid detection and / or sequencing methods disclosed herein can achieve high spatial isolation and concentration of microbial cells because, in stark contrast to liquid-phase methods in which microbial cells are spatially distributed and replicate throughout the entire liquid volume of the liquid growth medium, microbial cells are localized to small microcolony regions of the solid growth medium and replicate within these regions.

[0135] Microbial cells from microcolonies, through spatial isolation and concentration, can be efficiently recovered for nucleic acid detection and / or sequencing with high purity and isolation compared to the background, such as host cell debris and other interfering substances. The efficacy and practicality of this spatial isolation and concentration step can be easily understood and evaluated by comparing the generation and isolation achievable by microcolonies with the purification and isolation achieved by conventional centrifugation methods. For example, in a conventional centrifugation step, 1 mL of liquid sample can be treated by centrifugation to separate microbial cells, the supernatant can be removed, and a 100 μL microbial cell concentrate can remain. After adding 1 mL of dilution buffer, this step can be repeated, thereby concentrating the microbial cells tenfold and diluting the interfering substances tenfold. If two additional dilution and centrifugation steps are performed, the overall concentration factor of microbial cells remains at 10, while the dilution factor remains at 10. 3 It increases to that point.

[0136] This microcolony-based method allows for significantly higher resolution due to the spatial localization of growth and concentration. For example, consider an exemplary case where a 100 μL sample containing 10 CFU is grown in a solid growth medium with a diameter of 60 mm, the growth medium is incubated to support microcolony growth, and 10 microcolonies are formed. When the diameter of the microcolony is 50 μm, and a given microcolony is detected and efficiently recovered, the spatial concentration achieved is [(60 mm) / (0.05 mm)]. 2 ~10 6 Therefore, if microcolonies are collected with a collection diameter of 500 μm, the spatial concentration achieved is [(60 mm) / (0.5 mm)]. 2 ~10 4 This high degree of spatial concentration—and dilution of interferants—is significantly higher than that achievable with four rounds of centrifugation in previous examples. Furthermore, unlike conventional centrifugation approaches where some interferants continue to settle during subsequent rounds of washing and centrifugation, impairing the ability to achieve highly diluted factors, spatial isolation of microcolony localization and growth ensures highly purified factors upon recovery.

[0137] By combining both modalities, i.e., first performing centrifugal-based concentration (or another initial separation modality such as filtration or microfluidic separation) followed by microcolony growth, a remarkable degree of isolation and concentration can be achieved.

[0138] Furthermore, it should be noted that the method of this disclosure, which involves preparation from a direct sample by an intervening separation step of any microcolony, and in which nucleic acid amplification and / or sequencing are performed on the recovered microcolony, can be carried out without the need to divide the initial sample into multiple aliquots, as the infection detection and subsequent processing flow directly from the sample, the detected microcolony, and the recovery of microbial cells from the detected microcolony. This can interfere with the sensitivity of other approaches to direct processing of the sample that rely on sample division for initial infection detection and subsequent nucleic acid detection or AST.

[0139] This microcolony-based method is also advantageous when isolating microbial cells from antibiotics that may already be present in the initial sample. In fact, in the case of whole blood samples obtained from patients suspected of sepsis, it is common to initiate empirical antimicrobial treatment before initial venotomy. Although the initial isolation step to isolate microbial cells from the sample (e.g., centrifugation, filtration, and / or microfluidic separation) may result in a decrease in antimicrobial concentration, subsequently seeding the isolated microbial cells onto a solid growth medium can be effective in diluting the concentration of antimicrobial agents initially present in the sample by diffusing them into the solid growth medium, thereby reducing the local concentration of antimicrobial agents during microcolony growth.

[0140] Accordingly, various embodiments of this disclosure utilize microcolony-based growth and detection, as well as the recovery of microbial cells from detected microcolonies, while the colonies remain in the microcolony stage, to provide a purified and concentrated source of microbial cells for carrying out downstream assays and applications, such as rapid nucleic acid detection and / or sequencing for rapid detection of drug resistance markers.

[0141] Microcolony-based detection of resistance markers and rapid symptom identification panels. Figure 1A shows an exemplary method for performing microcolony-based nucleic acid detection. In step 100, the sample is processed according to the isolation method to separate microbial cells from the sample and obtain an initial suspension of microbial cells. In step 110, the suspension of microbial cells is brought into contact with a solid growth medium (examples are described below). As shown in step 120, the growth medium is incubated to support the growth of microcolonies and optically monitored by microscopic imaging for microcolony detection. In step 130, microcolonies are detected and located, as shown in the example below, which typically occurs within 1-3 hours of seeding in the solid growth medium. In step 140, microbial cells are recovered from the detected microcolonies (or multiple microcolonies) and resuspended to form a concentrated suspension (e.g., resuspended in a buffer). The recovery step schematically shown in Figure 1A can be carried out according to many different approaches, examples of which are detailed below.

[0142] A nucleic acid amplification assay is performed using recovered and resuspended microbial cells (by amplifying the nucleic acids released after lysis of the recovered microbial cells) to detect drug resistance genes associated with microbial cells in step 160, without further processing of the microbial cells after the recovery of the biological cells. A wide range of nucleic acid amplification assays can be performed to measure the presence or absence of at least one drug resistance gene. In some exemplary implementations, the nucleic acid amplification assay performed on recovered microbial cells may yield a single result, such as at least one specific target drug resistance gene, or multiple nucleic acid amplification assays may be performed to detect the presence of a panel of drug resistance genes. In some exemplary implementations, the drug resistance panel may target one or more resistance genes represented by Gram-positive pathogens, including, but not limited to, mecA, mecC, msrA, and erm genes (associated with Staphycoccus) and VanA and VanB genes associated with Enterococci, and / or, but not limited to, one or more resistance genes represented by Gram-negative pathogens, including CTX-M, IMP, KPC, NDM, OXA, and VIM genes (associated with Enterobacteriaceae, Pseudomonas, and Acinetobacter).

[0143] Figure 1A illustrates an exemplary method involving the detection of one or more drug resistance genes by performing one or more nucleic acid amplification assays on microbial cells recovered from microcolonies. In other exemplary embodiments, target nucleic acid sequences detected by nucleic acid amplification assays performed on microbial cells recovered from microcolonies can characterize aspects of the microbial cells other than the presence of drug resistance genes.

[0144] In some exemplary implementations, nucleic acid amplification assays performed on recovered microbial cells can provide single assay results, such as nucleic acid amplification assays that detect a specific drug resistance gene of interest, a specific microbial cell kingdom of interest, a specific microbial cell genus of interest, a specific microbial cell species of interest, a specific microbial cell line of interest, or a single Gram state of interest (with the presence or absence of other Gram states inferred).

[0145] The amplification product, often referred to as an amplicon, can be detected by methods known in the art, such as turbidity detection, visual detection of a binding probe, and fluorescence detection using an intercalation dye or fluorescent probe. In other exemplary methods, the amplification product may be separated by size using gel electrophoresis and visualized using a DNA intercalation dye such as ethidium bromide or SYBR Green. The size of the DNA fragment can be measured by comparing it to a DNA ladder.

[0146] Figure 1B illustrates an exemplary method involving the implementation of a panel of nucleic acid amplification assays based on microbial cells recovered from microcolonies. Thus, in some exemplary implementations, multiple nucleic acid amplification assays may be implemented based on recovered microbial cells as an assay panel (e.g., a symptomatic panel) configured for the detection of multiple microbial pathogens, and each nucleic acid amplification assay in the assay panel may be configured to detect different identification characteristics (or types) of microbial cells, such as, but not limited to, microbial cell kingdom, genus, species, strain, resistance markers, or Gram status (e.g.). In some exemplary implementations, the assay panel may be configured to detect and identify multiple microbial pathogens. In some exemplary implementations, the assay panel may be configured to detect and identify multiple microbial pathogens and multiple drug resistance genes. In some exemplary implementations, the assay panel may include one or more replication assays and / or one or more quality control assays. At least two nucleic acid assays in the assay panel may be spatially multiplexable as separate reactions. At least two nucleic acid assays in the assay panel may be multiplexed within a common reaction. At least two nucleic acid assays in the assay panel can be performed as nested nucleic acid amplification reactions.

[0147] A symptomatic panel may be configured to detect a set of pathogens associated with a clinical indication, such as, but not limited to, bloodstream infections (bacteremia), respiratory infections, urinary tract infections, vaginal and / or sexually transmitted infections, gastrointestinal infections, and drug resistance.

[0148] In several exemplary implementations, the recovered microbial cells are lysed, followed by a nucleic acid amplification reaction. As detailed below, the inventors have found that nucleic acids can be detected without problems from the recovered microcolonies using a variety of lysis methods. In particular, as described below, the inventors have found the surprising result that, contrary to conventional expectations in the art that thermal lysis is usually insufficient for lysing Gram-positive microbial cells, thermal lysis can be an effective lysis method for a wide range of Gram-negative and Gram-positive pathogens.

[0149] The inventors have discovered that, due to the high purity of the recovered microbial cells, after resuspending the recovered microbial cells in a buffer (compatible with nucleic acid amplification) and after lysing the recovered microbial cells in the buffer, the nucleic acids released from the microbial cells can be directly amplified without the need for nucleic acid extraction or isolation steps.

[0150] The inventors have also made the remarkable discovery that, in some cases, material from recovered microcolonies can be directly subjected to nucleic acid amplification in the absence of a microbial cell lysis step and a nucleic acid extraction or isolation step. While not intended to be constrained by theory, the microcolony matrix may contain nucleic acids that facilitate nucleic acid detection from recovered microcolonies even when there is no lysis of microbial cells within the microcolonies, and / or the proliferating microbial cells within the microcolonies may be more efficiently lysed during the thermal cycling of amplification reactions such as hot-start PCR, but not limited to these.

[0151] Sequence determination Although Figures 1A and 1B disclose a method for performing rapid nucleic acid detection based on microbial cells recovered from microcolonies, it will be understood that in some exemplary embodiments, microbial cells recovered from microcolonies may be used for sequencing, as shown in step 170 of Figure 2.

[0152] In some exemplary embodiments, after detection of microcolonies, microbial cells recovered from the microcolonies can be used for sequencing via an intermediate amplification step configured to increase the amount of nucleic acid to a level sufficient for initial sequencing. For example, whole-genome sequencing can be performed first to amplify the nucleic acid released from the recovered microbial cells. While not intended to be theoretically bound, the inventors believe that the purity of cells recovered from microcolonies can improve the fidelity of the resulting amplified nucleic acid in terms of the reduction in the relative amount of host DNA compared to other isolation modalities, thereby increasing the robustness of the overall sequencing method. Non-limiting methods for whole-genome amplification (WGA) include, but are not limited to, polymerase chain reaction (PCR)-based methods such as primer extension pre-amplification PCR (PEP-PCR), degenerate oligonucleotide-primer PCR (DOP-PCR), tagged random-primer PCR (T-PCR), LA-PCR (long and accurate PCR), ligation-mediated PCR (LM-PCR), and dispersed repeat-sequence PCR (IRS-PCR), as well as isothermal amplification methods such as multiple substitution amplification (MDA), multiple annealing and loop-forming amplification cycles (MALBAC), and linear amplification by transposon insertion (LIANTI).

[0153] In some exemplary embodiments involving the use of microbial cells recovered from microcolonies for sequencing, the relative purity of the recovered microbial cells may enable sequencing to be performed in the absence of additional steps used to enhance the amount of microbial cell nucleic acid compared to host genomic acid, in other cases. For example, in some exemplary embodiments, whole-genome amplification of nucleic acids obtained from recovered microcolonies can be performed in the absence of block primers. Furthermore, as will be further detailed below, the microcolony-based method of this embodiment enables sequencing-based detection of microbial cells without the need for splitting clinical samples.

[0154] As illustrated in the following examples, using microbial cells recovered from microcolonies addresses several key challenges in nucleic acid amplification and sequencing approaches to microbial cell detection. First, as mentioned above, the ability to rapidly and clearly detect microcolonies before amplification and / or sequencing is performed avoids the cost burden associated with the need to perform such assays on negative samples in other cases. Second, spatial purification associated with the localization, growth, and recovery of microcolonies substantially reduces or further avoids the challenges associated with debris and assay inhibitors, allowing the recovered microbial cells to be processed by nucleic acid amplification and sequencing assays with minimal or no nucleic acid extraction steps. Third, the increased microbial load during the initial growth phase before microcolony detection and recovery improves the sensitivity of the nucleic acid amplification assay and / or reduces the burden in sample preparation for sequencing applications.

[0155] In some exemplary implementations, cells recovered from microcolonies are analyzed by next-generation sequencing (NGS). Non-limiting examples of NGS include sequencing-synthesis, sequencing-ligation, sequencing-hybridization, and nanopore sequencing.

[0156] In some exemplary implementations, after resuspending the recovered microbial cells in a buffer and after lysing the recovered microbial cells in the buffer, the nucleic acids released from the microbial cells can be sequenced without requiring nucleic acid extraction or isolation steps.

[0157] Various exemplary steps of the methods disclosed in Figures 1A, 1B, and 2, and their variations, are described in detail in subsequent parts of this disclosure.

[0158] Seeding and incubation of solid growth media Microbial cells can be seeded on solid growth media according to a wide range of seeding methods. Non-limited seeding methods include spiral plating (e.g., distributing while rotating BD Kiestra® and bioMerieux WASP® plates, moving the distribution nozzle radially from the center to the periphery), mechanical diffusion (distributing the sample onto a plate and distributing it by rods, loops, or beads), spin coating, and distribution in the form of droplets, typically having a volume of 10 μL. Alternatively, a combination of the above methods can be used. In one exemplary implementation, the seeding method is selected so as to minimize the preferential accumulation of debris and other contents (commonly known as the coffee stain effect) during liquid drying. In another case, an opaque layer may cover the microbial cells, thereby affecting the growth of microbial cells and microcolony monitoring.

[0159] In some exemplary methods, a sample suspected to contain microbial cells can be directly seeded into a solid growth medium. In other exemplary methods, the sample suspected to contain microbial cells can be treated before seeding into the solid growth medium to isolate and selectively concentrate the microbial cells from the sample, thereby providing an initial suspension of microbial cells for seeding into the solid growth medium. Generally, the degree to which the sample is treated beforehand depends on both the nature of the sample and the degree of purification / dilution of assay interferants within the sample.

[0160] By diffusing / distributing a suspension of microbial cells onto the surface of a solid growth medium, concentrated debris and interfering substances are reduced, and the ratio of microorganisms to human cells or nucleic acids present in the sample is improved. An exemplary method of diffusion is disclosed in Example 5.

[0161] The seeded solid growth medium is incubated at an appropriate temperature and environment to promote microcolony growth. For example, after seeding, the solid growth medium can be incubated within a controlled temperature range, such as 35-38°C, and a controlled humidity, such as saturated humidity. Examples of suitable solid growth media are detailed below.

[0162] Microcolony monitoring and detection After distributing isolated microbial cells onto a solid growth medium, the growth of microcolonies formed on the solid growth medium can be intermittently monitored according to one or more detection modalities. Monitoring can be performed in situ within the incubation environment, or by temporarily removing the solid growth medium from the incubation environment, performing microcolony detection measurements, and then returning the solid growth medium to the incubation environment.

[0163] In one exemplary implementation, optical detection can be used to monitor the growth of one or more microbial cell colonies. For example, in one exemplary implementation, a camera can be used to image at least a portion of a solid growth medium. In another exemplary implementation, an image of one or more microbial cell colonies can be obtained using an array of photodetectors in the absence of an imaging element, provided that the growth surface associated with the colonies is located close enough to the array of photodetectors and the photodetectors form an image when the array is illuminated. In implementations where the field of view of the optical system is less than the spatial extent of the solid growth medium, the optical system can be scanned over the solid growth medium or vice versa to facilitate optical inspection of the entire surface of the solid growth medium or a desired subset thereof. Exemplary methods for microcolony monitoring are disclosed in international patent applications PCT / CA2019 / 051895 and PCT / CA2021 / 050884.

[0164] Images can be processed using known image processing algorithms to identify microcolonies, and one or more dimensional measurements of the identified microcolonies can be optionally estimated. For example, commonly available software such as the ImageJ / Fiji program can be used. In one exemplary method, the image can be converted to a grayscale image, and then the grayscale image can be binarized by applying locally applicable image thresholding according to the Phansalkar method based on histogram analysis of intensity levels. Applicable image segmentation can then be used according to the Phansalkar method, with dimensional constraints on dividing the image into segments for microcolony identification. Then, optional optical analysis of the identified segments can be used to measure the relevant metrics (e.g., roundness, area, major axis, and minor axis) associated with the microcolonies. Many different exemplary algorithms exist that can be used to calculate thresholds without bias. The Phansalkar thresholding method is an improved version of the Sauvola thresholding method optimized for low-contrast images [Phansalskar, N; More, S & Sabale, A et al. (2011), “Adaptive local thresholding for detection of nuclei in diversity stained cytology images.”, International Conference on Communications and Signal Processing (ICCSP):218-220, doi:10.1109 / ICCSP.2011.5739305]. Other exemplary methods include the Bernsen, Contrast, Mean, Median, MidGrey, Niblack, Otsu, and Sauvola methods. Image registration can be performed using a variety of registration methods, including, but not limited to, feature-based, intensity-based, and non-rigid registration algorithms.Examples of suitable feature-based algorithms include SURF (Speeded Up Robust Features) and SIFT (Scale Invariant Feature Transformation).

[0165] In one exemplary implementation, image registration can be performed by an intensity-based algorithm as follows: The algorithm transforms moving images (images acquired at a later point in time) to be spatially registered together with static / reference images (images acquired at a later point in time). Depending on the setup, the type of transformation performed may be defined as "rigid" or "affine". According to the simplified definition, the algorithm internally constructs a multi-resolution pyramid in memory (at user-specified pyramid levels) and solves an optimization problem at each level of the pyramid. In other words, the algorithm constructs an image pyramid with N levels (e.g., N=5). At each pyramid level, the image dimensions are reduced by a factor of two. Optimization begins at the coarsest level of the pyramid and continues until the user-allowed number of iterations is reached, or until the optimizer converges on an attempt to refine the current transformation estimation at subsequent pyramid levels.

[0166] Alternatively, other image alignment techniques such as SIFT, BRIEF, ORB, and AKAZE can be used. Furthermore, if features are reduced due to blurring caused by excessive debris, it is preferable to perform feature extraction in the frequency domain. An exemplary implementation is shown in Example 4C.

[0167] The time-lapse imaging method in this embodiment is a semi-quantitative imaging technique that captures a series of images of the same (or nearly the same) scene at different points in time, capturing dynamic changes while classifying and removing static components as background. Current approaches to dynamic profiling of microcolonies rely on estimations of static background and illumination. However, such techniques can be subjected to numerous processing variations when the spectral or spatial features of the solid growth medium debris or surface are not static. First, it should be noted that a suitable environment must be provided during image acquisition that allows the microcolonies to survive without the surface substantially aging (changes in debris size and evaporation of liquid from the solid growth medium surface associated with debris replacement).

[0168] The measurement of colony cell content (e.g., whether there is a sufficient colony size and / or cell number for nucleic acid amplification and / or sequencing) can be achieved by various approaches. In one exemplary implementation, one or more geometric or optical properties associated with the colony (not limited to radius / area or scattering / reflection / transmission intensity, measured from image processing methods such as image segmentation) can be processed to determine whether there is a sufficient number of microbial cells in the colony for subsequent processing, based on a comparison of one or more geometric properties with reference data relating to the number of microbial cells. In another embodiment, a neural network can be used to measure a given number of microbial cells in the colony. Based on the imaging, it is possible to measure whether a sufficient number of microbial cells are present in a colony for subsequent testing (or, for example, for subsequent testing of a given type, for a known binary measure of whether a sufficient number of microbial cells are present in the colony), where a neural network is trained on images of a reference strain with a known number of cells. In some exemplary implementations, the measurement of whether a sufficient number of microbial cells are present in a given colony can be done in part on the detected or inferred identity of one or more taxonomic classes (e.g., Gram status, genus, family, species, strain, etc.) of the colony.

[0169] Optical imaging is just one example of a detection modality for monitoring microbial cell growth; it should be understood that other detection modalities, such as electrical impedance analysis, could be used as alternatives to detect volatile organic compounds associated with microbial cell growth or to employ calorimetry.

[0170] Exemplary experimental measurements of the correlation between microcolony size and microbial cell number were performed according to Examples 1-6 (described below). To determine the average time to positivity, microbial cell suspensions containing approximately 100 CFU of various microbial cell species were seeded on solid growth medium, incubated, and intermittent microscopic imaging was performed. The "time to detection" (TTD) was defined as the time at which no new microcolonies were detected even after further incubation. The measurement times for detecting different bacterial cells are shown in Figure 3. Furthermore, the average diameter of microcolonies after 4 hours of incubation is included in the table.

[0171] Microcolony detection and size thresholding for nucleic acid amplification In all microbial species tested to date, the inventors have found that by the time microcolonies can be detected with a diameter of 20–50 microns by microscopy, they have a large number of microbial cells, facilitating nucleic acid amplification after recovery (positive results are detectable when the diameter is approximately 20 microns in a whole blood cell suspension, and the inventors have found that microbial cells recovered from such small microcolonies surprisingly have sufficient biomass in molecular amplification assays). Therefore, in some exemplary embodiments, once microcolonies are detected, they can be considered ready for recovery.

[0172] In one exemplary embodiment involving optical imaging of microcolonies grown in solid growth medium, the colony dimensions or size, measured directly or indirectly, can be optionally used, along with more properties of a given imaged colony (such as the type of microbial cells in the colony, e.g., genus, family, species, or strain), to estimate the number of microbial cells in the colony and / or determine the time at which incubation can be completed and the time at which the microcolonies can be harvested for nucleic acid amplification and / or sequencing. Determining the appropriate growth time to achieve a desired number of microbial cells may vary depending on the cell type (e.g., genus, family, species, strain).

[0173] The relationship between microbial cells and the time it takes to reach a sufficient number of cells for subsequent nucleic acid amplification can be established according to a lookup table. For example, an automated system can use optical image processing to measure the cell class associated with a given colony (e.g., an inferred or estimated microbial species), and then, using a predetermined relationship (e.g., a lookup table or a predetermined functional relationship), measure, for example, the suitable time for a sufficient number of microbial cells to be present in one or more colonies for subsequent processing, or a suitable size indicator (e.g., radius or other spatial indicator) for a sufficient number of microbial cells to be present in one or more colonies for subsequent processing. In some exemplary embodiments, multiple criteria, including both colony size indicators and time, can be correlated with the microbial cell class to estimate when a sufficient number of microbial cells are present in a colony.

[0174] Microcolony detection and size threshold for sequencing In some exemplary embodiments involving sequencing, detected microcolonies can be monitored until their size is considered sufficiently large for sequencing without the initial amplification step before library preparation. For example, experimental studies can be conducted to measure the relationship between colony size and quantity of a given type of nucleic acid (e.g., genomic DNA), thereby determining a given sequencing biomass limit (e.g., 50 ng, 100 ng, or 200 ng) or a given sequencing CFU limit (e.g., 10 6 Or 10 7 This enables the measurement of a suitable colony size threshold for individual cells. This threshold may be optionally measured at the species (or other suitable taxonomic rank) level, which is initially determined by estimation, for example, by optical microscopy imaging disclosed in International Patent Application PCT / CA2021 / 050884, or by recovering microbial cells from a second growing microcolony and performing a multiplexed nucleic acid amplification identification panel, according to the exemplary embodiments or variations thereof described above.

[0175] After colonies have grown beyond the microcolony stage (e.g., having a diameter greater than 100 μm), a threshold for biomass abundance for sequencing library preparation without initial amplification may arise for at least some types of microbial cells. Therefore, in some exemplary implementations, microcolonies can be collected for sequencing during the microcolony stage (e.g., when the first amplification is performed), while in other exemplary implementations, colonies that are no longer microcolonies can be collected for sequencing after, for example, the colony diameter (or effective diameter) is greater than 100 μm but less than 150 μm, or after the colony diameter is greater than 100 μm but less than 200 μm, or after the colony diameter is greater than 100 μm but less than 250 μm, or after the colony diameter is greater than 100 μm but less than 300 μm, or after the colony diameter is greater than 100 μm but less than 350 μm, or after the colony diameter is greater than 100 μm but less than 400 μm, or after the colony diameter is greater than 100 μm but less than 450 μm, or after the colony diameter is greater than 100 μm but less than 500 μm.

[0176] collect Microbial cells can be recovered from solid growth media for any downstream assay, application, or purpose according to a wide range of recovery methods. Recovery may be automated, manual, or semi-automatic. Using the detected location of a given microcolony, recovery may be facilitated by, for example, automated commands (e.g., a robotic recovery step) or by indicating the microcolony location (e.g., by annotation on a display showing illumination or images). The recovered microbial cells may be processed to provide a form suitable for downstream applications such as nucleic acid amplification, sequencing, MALDI-based identification, Raman spectroscopy, and other identification modalities such as antimicrobial susceptibility testing. In some exemplary implementations, the recovered microbial cells may be diluted or concentrated. In some exemplary implementations, the recovered microbial cells may be combined with a liquid to form a suspension, which may be optionally diluted or concentrated and optionally aliquoted before carrying out one or more downstream applications such as nucleic acid amplification assays or sequencing methods.

[0177] Various non-limiting, exemplary methods for recovering microcolonies are described in the following chapters and in Example 8 below. It will be understood that different recovery methods may be appropriate, advantageous, or preferred for different applications, for example, depending on the ease of implementation, the ability to perform localized recovery (and avoid interference with microcolonies adjacent to the target microcolony), and the required or desired recovery efficiency.

[0178] In one exemplary embodiment, once the colony's location is identified, it can be manually collected using a biopsy punch, seeding loop, or sterile cotton swab. In several other exemplary embodiments, once the colony's location is identified, it can be collected by a robot.

[0179] For example, a circular instrument can be used to collect small sections of solid growth medium from microcolonies, similar to a biopsy punch. U.S. Patent Publication No. 2018 / 0284146 describes a device comprising a platform for holding a culture plate and a movable robotic arm having a pinching tool that can be lowered to pick up colonies from the plate. Another part of the device houses a sterile tube containing a suspension medium. After pinching a portion of the colony, the pinching tool moves to transfer the pinched colony into the sterile tube. Optionally, the tool may include an ultrasonic transducer or vortex mixer for more efficient release of the collected microbial cells. In some exemplary implementations, a pipette tip can be used as a biopsy device for removing sections of culture medium containing microcolonies.

[0180] Another exemplary method for collecting microcolonies is the scraping method. This method involves scraping bacteria from a solid growth medium using a sterile tool (seeding loop or spatula). The tool is then immersed in a tube containing a clean buffer (e.g., phosphate buffer) and stirred to release the collected cells.

[0181] Another exemplary microcolony recovery method is a wash recovery method in which microbial cells are removed from the gel surface along with a buffer (preferably a buffer compatible with downstream assays or other downstream applications). In some exemplary implementations of the wash method, the buffer is distributed to the microcolonies on the gel surface, and after distribution, the microcolony surface is mechanically disrupted (e.g., selectively scraped using a distribution device) so that the microbial cells become a suspension in the buffer. The suspension is then stirred to remove the microbial cells. An exemplary evaluation of the performance of the wash method is described in Example 8B. Figure 4A shows the number of colonies after recovery of microcolonies of various bacterial cells after 4 hours of growth following seeding. Here, it is shown that a large number of microbial cells were recovered by the recovery method after only 4 hours of microcolony growth. The number of cells in each recovered microcolony corresponds to the average of 5 microcolonies, and recovery was performed using the “wash” method described in Example 8B. The inventors also found that similar cell counts were obtained when recovery was repeated using the other three methods in Example 8, similar to those in this example using the wash method.

[0182] In some exemplary implementations, retrieval is performed by bringing microcolonies into contact with a surface treated to exhibit affinity for microbial cell retrieval / capture; such a surface is hereafter referred to as an "affinity surface." In some exemplary embodiments, retrieval by contact with the affinity surface can be performed by gently bringing a retrieval chip into contact with the microcolonies, transferring the microbial cells to the chip, and fixing the cells by a force such as electroattraction. A retrieval chip having an affinity surface may be, for example, a flat surface (affinity surface) that is substantially larger than the area of ​​the microcolonies and has affinity for microbial cell collection / capture.

[0183] The area of the affinity surface can be selected taking into account the placement tolerance of the recovery device such that it can spatially enclose the microcolonies or based on the user tolerance measured when performing manual recovery. However, in order to avoid the movement of an excessive amount of residual debris with the microbial cells in the solid growth medium or to avoid the movement of an excessive amount of growth medium as part of the recovered sample, it may be beneficial to limit the size (spatial extent) of the affinity surface relative to the size of the recovered microcolonies. Limiting the size of the affinity surface may also be beneficial to assist in ensuring sufficient contact between the affinity surface and the microcolonies when the affinity surface is precisely parallel to the surface of the solid growth medium. In some exemplary embodiments, the area of the affinity surface is less than 10 mm 2 less than 5 mm 2 less than 4 mm 2 less than 3 mm 2 less than 2 mm 2 less than 1 mm 2 less than 0.5 mm 2 less than 0.1 mm 2 less than and 0.05 mm 2 and is. In some exemplary embodiments, the area of the affinity surface is 1 mm 2 ~10 mm 2 1 mm 2 ~5 mm 2 1 mm 2 ~4 mm 2 1 mm 2 ~3 mm 2 0.5 mm 2 ~2 mm 2 0.1 mm 2 ~1 mm 2 0.0.5 mm 2 ~0.5 mm 2 ~0.05 mm 2 ~0.1 mm 2 and is.

[0184] In some exemplary implementations, the affinity surface may be chemically treated. In other exemplary embodiments, the affinity surface may be coated with an adhesive material. In some exemplary embodiments, the adhesive material may be coated onto the chemically treated surface. Affinity-assisted retrieval of microbial cells, which moves to the affinity surface, can be facilitated, for example, by nonspecific adsorption and / or physical uptake of microbial cells on the surface.

[0185] Examples of suitable coating surfaces for forming affinity surfaces include Fisherbrand® Superfrost® Plus Microscope Slides (Control SF), PDL (poly-D-lysine), PLL (poly-L-lysine), Rat tail Collagen I (collagen), mouse laminin (laminin), fibronectin on a PDL layer (fibronectin), and gelatin from Neurovitro Corporation or positively charged slides from Walter Products Inc.

[0186] Another example of a recovery method is the aspiration method, which is carried out as follows: The recovery buffer is drawn into a pipette tip or another suitable fluid distribution device, such as a fluid transfer tube connected to a pump or aspiration device (commonly used in automated fluid handling systems, for example). The tip is moved over the microcolony and pointed downwards, bringing it into contact with the gel surface around the microcolony, thereby forming a fluid seal between the tip and the gel, trapping the microcolony without damaging the gel. The recovery buffer is then distributed onto or into the gel, allowing at least a portion of the recovery liquid to permeate the gel surface. The distributed recovery buffer is then aspirated, attracting the microbial cells released from the microcolony to the tip, forming an aspirated suspension. The recovery suspension containing the microbial cells can then be distributed to an optimal location or container.

[0187] The aspiration method is schematically represented in Figure 28A. As can be seen in the figure, a seal is formed between the distal open end of the tip and the surface of the gel, allowing the recovered buffer to penetrate at least partially into the gel during the initial dispensing operation. In some exemplary implementations, the tip is pushed into the gel, and the distal end of the tip permeates through the gel and extends into the gel, thereby increasing the effectiveness of the seal and potentially improving the ability of the recovered buffer to permeate through the gel and extend into the gel during the dispensing operation.

[0188] The inventors discovered that the efficiency of microcolony collection by aspiration depends on the position of the distal end of the collection tip relative to the gel surface. To demonstrate and investigate this dependence, microcolonies of Staphylococcus aureus ATCC 29213 were collected after being grown on blood agar gel at 37°C for 5 hours. In step 6, the collection was carried out according to the procedure described in Example 8D, except that the distance of the tip from the gel surface was set as a free parameter. The collection process was repeated three times at each height, and serial dilutions of the collected microcolonies were incubated overnight for biomass measurement.

[0189] Figure 28C shows the average recovered biomass as a function of tip height. As can be seen, the recovery efficiency is maximized in the range of [-0.6 mm, -0.2 mm]. This represents the advantage of sealing the tip-gel interface without disrupting the gel. It is understood that, with sufficient precision in placement, the work can be completed by reducing it to 0.1 mm or even less. Thus, in some exemplary embodiments, the suction recovery method can be used by introducing the distal end of the tip into and below the gel surface in amounts ranging from 0 to 0.2 mm, 0 to 0.4 mm, 0 to 0.6 mm, 0 to 0.8 mm, 0 to 1.0 mm or, for example, 0.1 to 0.2 mm, 0.1 to 0.4 mm, 0.1 to 0.6 mm, 0.1 to 0.8 mm and 0.1 to 1.0 mm or, for example, 0.2 to 0.4 mm, 0.2 to 0.6 mm, 0.2 to 0.8 mm and 0.2 to 1.0 mm.

[0190] The inventors have found that the aspiration method is efficient, easy to monitor, and allows for the localization of microcolonies during collection and concentration of the collected cells, which may be beneficial when it is desired to suspend microbial cells in a small volume of liquid (e.g., a resuspension suspension).

[0191] To demonstrate the reliability of the aspiration method for recovery, the experiment was conducted according to the method of Example 8D. In this case, the tip was lowered by 0.3 mm after contact with the gel surface. The measured recovered biomass, averaged across three microcolonies for various pathogenic bacterial cells, is shown in Figure 28D. As observed, the data demonstrates the good repeatability of the microcolony recovery approach, considering the fact that the observed coefficient of variation (CV) values ​​can be easily justified by the expected variation in growth rates from one microcolony to another.

[0192] It will be understood that the height of the distal end of the tip relative to the gel surface can be controlled by many different exemplary methods and / or mechanisms, for example, to ensure a predetermined insertion depth of the distal end of the tip relative to the gel surface. For example, if the gel surface is known to have a constant height within various gel supports used for microcolony growth, the tip can be positioned in an open-loop configuration relative to the gel using an initial calibration of the tip height and the gel surface. In another embodiment, a closed-loop configuration can be used, for example, by using feedback from a conductive pipette tip or feedback from a depth sensor, or a force sensor, or a pressure sensor, to enable measurement of contact with the gel surface (as described, for example, in Example 8D).

[0193] The inventors have found that this recovery method and the exemplary implementation described in Example 8 below are suitable for performing downstream applications such as extraction of microbial cells and nucleic acid amplification and / or sequencing, as shown below. However, in some cases, the recovery process may cause localized damage to the solid growth medium, hindering further monitoring of any residual cells along with any unrecovered portions of microcolonies.

[0194] In recovery methods other than biopsy, the local surface of the solid growth medium is less likely to be damaged during recovery, making it possible to continue monitoring the growth of residual microbial cells after recovery. Furthermore, as taught by the method of Example 3, the possibility of damaging the gel surface can be further reduced by using higher gel concentrations. In one embodiment, the gel concentration is selected to be 1.35% to 2.0% or 1.35% to 2.5%. In another embodiment, the gel concentration is selected to be 1.5% to 2.0% or 1.5% to 2.5%. In some applications, but not limited to, it may be useful to further grow microcolonies after recovery for nucleic acid amplification or sequencing to provide microbial cells for other downstream applications, such as identification by MALDI and / or antimicrobial susceptibility testing (AST).

[0195] Furthermore, in some applications, resuspending microcolonies in liquid culture media may be undesirable. For example, during tests such as microbial identification by MALDI, the distributed cell suspension may be affected by the "coffee stain effect" during drying, resulting in a more heterogeneous spatial distribution of cell content than the resulting stain. This can degrade the modality's performance. Microcolony recovery by affinity capture may be beneficial in mitigating these challenges.

[0196] In another exemplary embodiment, microcolony harvesting is performed using electrical force. The method of this embodiment is shown in Figure 4B and will be described later. The bacterial cell wall and membrane contain numerous proteins, lipid molecules, teichoic acid, and lipopolysaccharides that contribute to the characteristic negative charge at pH values ​​greater than 4. Therefore, for example, in the presence of an electric field generated near an electrode, bacterial cells are pulled towards positive polarity.

[0197] During the microcolony picking step of the recovery operation, preferably an electrically separated distal tip (e.g., a pin with an electrical separation layer formed on or provided on an electrode) is provided, with positive polarity (V gA slender electrode to which a saturation agent has been applied is brought near the microcolony. The electrode may have a flat distal tip (to which an electrical insulating layer is formed or applied) with a horizontal cross-sectional dimension of approximately 1 mm or less.

[0198] During the release phase, the polarity of the electrodes is negative (V) relative to the substrate to which the microcolonies are distributed. s ) and the recovered bacterial cells are released. V g and V s The scale of both can depend on several factors, such as the microbial cell species, the shape of the electrode, and the culture medium from which the microcolonies are harvested.

[0199] The inventors have found that voltages in the range of 3 to 50 volts may be suitable for efficient microbial cell migration (higher voltages are optional if damage can be avoided). A thin layer of dielectric material can be used for separation. In one implementation, a 1 Mil Kapton® HN film containing silicone is used. In another implementation, the electrode is aluminum and the separation layer is aluminum oxide produced by an anodizing procedure. Although contact with the substrate appears to be beneficial for achieving efficient electrostatic microbial cell migration, in some exemplary implementations, one or both of the collection and release operations can be performed without contact with the substrate, provided that the distal tip of the separated electrode is brought close enough to the substrate.

[0200] To demonstrate the implementation of electrostatic microcolony recovery, 4-hour-old microcolonies were prepared from exemplary Gram-negative bacteria (Escherichia coli) and exemplary Gram-positive bacteria (Enterococcus). Five microcolonies of each bacterium were then recovered using the aspiration method of Example 8D as a reference.

[0201] Although an exemplary electrostatic microcolony recovery method has been described and illustrated based on the recovery of bacterial microbial colonies, it will be understood that this electrostatic method is also applicable to and / or suitable for the recovery of fungal microcolonies.

[0202] While many of these exemplary embodiments focus on the recovery of a single microcolony, it will be understood that multiple microcolonies can be recovered, and microbial cells derived from multiple microcolonies can be processed, for example, by nucleic acid amplification, or sequencing. In some exemplary implementations, microbial cells derived from multiple microcolonies can be processed together to yield, for example, a larger biomass. For example, microbial cells recovered from multiple microcolonies can be combined (pooled) to yield sufficient biomass for a given downstream application, such as sequencing, with or without amplification before library preparation.

[0203] In other exemplary implementations, microbial cells from different microcolonies that have been recovered can be treated separately, for example, by nucleic acid amplification and / or sequencing, to yield microcolony-specific results. Such microcolony-specific treatment may be useful for detecting and identifying differences in cell type among multiple microcolonies, for example, in polymicrobial infections or by contaminating pathogens. Microcolony-specific treatment may also be useful for detecting heterotolerance among multiple growing colonies.

[0204] Based on imaging of microcolonies before recovery, the selection of a given microcolony for recovery can be determined. Based on the properties or characteristics of the microcolony measured by imaging before recovery, the selection of a given microcolony for recovery can be determined. Furthermore, the selection of a given microcolony recovery method and / or one or more parameters of a given microcolony method (e.g., a suitable recovery buffer, a suitable volume of recovery buffer, a suitable electrical recovery voltage, a suitable contact time interval, an appropriate time for distributing the recovery buffer and aspirating the suspension containing free microbial cells derived from the microcolony, and / or a suitable tip depth for aspirated recovery) may be made based on the properties or characteristics of a given microcolony (e.g., a class or classification of the microcolony type based on a machine learning algorithm for processing microcolony images).

[0205] In some exemplary embodiments, a phenotypic correspondence can be established between a first and a second colony before microbial cells are recovered from the second microcolony. This phenotypic correspondence can be established, for example, by comparing the two microcolonies with their associated classes or by comparing optical images or light signals detected from the two microcolonies. For example, a phenotypic correspondence can be established by a machine learning algorithm that classifies the two microcolonies as elements of a common class based on images of the microcolonies. As described in International Patents PCT / CA2019 / 051895 and PCT / CA2021 / 050884, the absence of a detected phenotypic correspondence can be used to identify polymicrobial colonies.

[0206] sample The methods of this disclosure can be used to process a wide variety of samples, including but not limited to urine, lymph, cerebrospinal fluid, blood (e.g., whole blood, blood cultures, and plasma), sputum, mucus, wound drainage, saliva, lavage fluid, joint fluid, abscess fluid, homogenized tissue suspensions (including, but not limited to, homogenized suspensions of feces, muscle tissue, brain tissue, and liver tissue), and any fluid aspirates or tissue extracts of human and / or other mammalian organs.

[0207] In some embodiments, the clinical sample contains 0.1 to 10 microbial genomes / milliliter, or 0.1 to 10 2 Microbial genome / milliliter, or 0.1-10 3 This includes the amount of DNA present in microbial genomes / milliliter. In some embodiments, the clinical sample is 0.1-10 CFU / mL, or 0.1-10 2 CFU / mL, or 0.1-10 3 This includes the amount of microbial cells present in CFU / mL.

[0208] In some cases, the sample may be substantially free of debris and potential interferences, facilitating direct seeding of solid growth media. For example, this may be achievable with sample types such as urine, but is not limited to these.

[0209] Initial isolation of microbial cells from samples before seeding in solid growth media. In many cases, a sample matrix and / or sample volume may be necessary to isolate and / or concentrate microbial cells before seeding onto solid growth media. This initial processing process may include the lysis of host cells, such as host blood cells. For example, after a whole blood sample, following initial lysis of host erythrocytes, one or more of numerous microbial cell isolation techniques can be used to provide a suspension of microbial cells suitable for seeding onto solid growth media. Non-limiting examples of such methods include lysis-centrifugation, filtration, and microfluidic flow-based separation.

[0210] Factors that may limit the detection of microcolonies are the size and density of surface artifacts (background) observable by microscope after the treated sample has been brought into contact with the solid growth medium, and such artifacts are not examples of microbial cells or microbial cell colonies. Two exemplary types of such surface artifacts that contribute to the background are (i) surface heterogeneity of the gel surface, and (i) dissolved debris particles that remain after sample dissolution, such as dissolved debris particles that are introduced by the digestion of blood cells and are present in the sample after centrifugation washing.

[0211] The surface density of the first type of artifact, i.e., the inhomogeneities on the gel surface, can be reduced by controlling gel formation. Exemplary non-limiting methods for preparing gels with low density of surface inhomogeneities are described in Example 3 below.

[0212] The density of the second type of artifact, i.e., debris particles derived from the sample, can be reduced by using appropriate methods for suitable initial host cell lysis and microbial cell separation. Various examples of such methods will be described later.

[0213] A blood lysis reagent for appropriately digesting and reducing surface debris In the case of blood, the inventors have discovered that it may be important to ensure sufficient lysis and digestion of host blood cells in order to obtain a microbial cell suspension (e.g., after centrifugation or filtration) that results in a sufficiently low spatial density of surface artifacts after seeding on a solid growth medium. It has been discovered that the size and amount of the second type of artifact, i.e., lysed debris particles, can vary between whole blood samples and depend on the composition of the blood lysis reagent (BLR).

[0214] Examples of blood lysis reagents for separating microbial cells from whole blood samples are disclosed in the specification of international patent application PCT / CA2013 / 000992 entitled "APPARATUS AND METHOD FOR EXTRACTING MICROBIAL CELLS" and in the specification of international patent PCT / CA2019 / 050716 entitled "METHODS AND COMPOSITIONS FOR THE SELECTIVE LYSIS OF BLOOD CELLS AND SEPARATION OF MICROBIAL CELLS", the latter of which is hereby incorporated by reference in its entirety.

[0215] International patent application PCT / CA2013 / 000992 discloses a number of different hemolytic reagent compositions that can be used to digest blood components before centrifugation. As described above, the presence of the hemolytic reagent induces the selective lysis of blood cells. In one exemplary implementation taught by international patent application PCT / CA2013 / 000992, the hemolytic reagent may be an aqueous liquid containing a saponin and sodium polyanetholesulfonate (sodium salt of polyanetholesulfonic acid, known as SPS), and a hemolytic reagent having such a composition will hereafter be referred to as a “Type 1” hemolytic reagent. The hemolytic reagent may also contain an antifoaming agent such as poly(propylene glycol) (e.g., PPG having a molecular weight of approximately 2000). International patent application PCT / CA2013 / 000992 teaches exemplary concentration ranges of saponins and SPS in type 1 hemolytic reagents, approximately 1.5–80 mg / mL and 0.5–20 mg / mL, respectively, when whole blood and hemolytic reagents are mixed.

[0216] As taught in international patent application PCT / CA2013 / 000992, SPS is an anticoagulant and phagocytosis inhibitor and is known to interfere with antimicrobial agents (Sullivan, NM, Sutter, VL, & Finegold, SM (1975). Practical aerobic membrane filtration blood culture technique: development of procedure. Journal of clinical microbiology, 1(1), 30-36). The mechanism by which SPS assists hematopoietic cell lysis is not fully understood. While not intended to be limited by theory, it is thought that SPS may, to some extent, protect microorganisms during hematopoietic cell lysis, reduce the occurrence of bacterial enclosure within cell debris, and / or, in other cases, reduce the amount of lysis debris components that may be present in the precipitate.

[0217] In another exemplary implementation of the hemolytic reagent composition taught by international patent application PCT / CA2013 / 000992, the hemolytic reagent may be an aqueous liquid containing Triton X-100 and SPS in a buffer having a pH in the range of 9 to 11, and a hemolytic reagent having such a composition will hereafter be referred to as a “type 2” hemolytic reagent. The hemolytic reagent may also contain an antifoaming agent such as poly(propylene glycol) (e.g., PPG having a molecular weight of approximately 2000). International patent application PCT / CA2013 / 000992 teaches exemplary concentration ranges of Triton X-100 and SPS for a type 2 hemolytic reagent, approximately 0.5 to 1.5% w / v and 5 to 10 mg / mL, respectively, when mixed with whole blood.

[0218] As described above, the type 1 hemolytic reagent composition described above has been found to be suitable for manual and semi-automatic separation and concentration of microbial cells derived from whole blood, as taught in international patent application PCT / CA2013 / 000992. However, when adapting the reagent formulation disclosed in international patent application PCT / CA2013 / 000992 for automated separation and concentration and subsequent identification of microbial cells derived from whole blood, according to the automated method described in international patent application PCT / CA2015 / 050449, filed on May 19, 2015, titled "Apparatus, System and Method for Performing Automated Centrifugal Separation," which is incorporated herein by reference in its entirety, the inventors have found that the type 1 hemolytic reagent composition is most suitable when the volume of whole blood is approximately less than 1 mL.

[0219] Another exemplary hemolytic reagent for achieving a low surface density of dissolved debris particles is described in international patent application PCT / CA2019 / 050716, which describes exemplary hemolytic reagent compositions and methods for protecting microbial cells and reducing sample viscosity. This hemolytic reagent composition, hereafter referred to as a type 3 hemolytic reagent, can be provided, containing a saponin, SPS, an alkaline buffer, and optionally a nonionic surfactant.

[0220] In one exemplary embodiment, the type 3 hemolytic reagent may have a composition such that, after mixing the hemolytic reagent with a sample, the saponin concentration is 3–60 mg / mL, the SPS concentration is 1.5–50 mg / mL, the nonionic surfactant concentration is 0–3% w / v, and the pH is 7.8–10. In some exemplary embodiments, the buffer concentration can be selected so that the effective buffer concentration is in the range of 10–300 mM. It will be understood that, but not limited to, the appropriate concentration range of a given component of the hemolytic reagent for a given set of conditions can be determined by experimentally investigating the effect of changes in the concentration of a given component on one or more performance metrics, such as hemolytic efficiency, amount of residual blood cell debris, integrity of microbial cells, and microbial cell viability. The type 3 hemolytic reagent may be provided as two or more reagents that can be stored separately and mixed before use, and the saponin component of the hemolytic reagent is stored in an acidic environment separate from the alkaline component of the hemolytic reagent. In one exemplary implementation, one or more reagents that are mixed to form the final hemolytic reagent may be stored in a solid phase.

[0221] Therefore, in some exemplary implementations, samples can be treated with a Type 3 lysis reagent having an alkaline pH, comprising saponin, sodium polyanethole sulfonate (SPS), a nonionic surfactant (but not limited to Triton® X-100), and a buffer (e.g., carbonate-bicarbonate buffer). The blood lysis reagent may also include an antifoaming agent such as SE-15 (e.g., a 10% w / v emulsion of an active silicone polymer and a nonionic emulsifier).

[0222] Hemolytic reagents that do not contain nonionic surfactants and carbonate-bicarbonate buffers are known to be benign to microbial cells. However, as described below, when processing whole blood samples, the size of the blood debris transferred to the final cell suspension has been found to increase the level of surface artifacts (background) as a result of using such hemolytic reagents, which can hinder microcolony detection and characterization. In contrast, the addition of appropriate amounts of nonionic surfactants (e.g., Triton® X-100) and carbonate-bicarbonate buffers along with saponins and SPS may be beneficial in that it significantly reduces surface artifacts generated from soluble debris without significantly affecting cell viability.

[0223] In some exemplary implementations, a whole blood sample of up to 10 mL can be mixed with the type 3 hemolytic reagent such that the saponin concentration in the final mixture is in the range of 10–30 mg / mL (or 3–60 mg / mL in some exemplary implementations), the SPS concentration in the final mixture may be in the range of 5–50 mg / mL (or 1.5–50 mg / mL in some exemplary implementations), the effective buffer concentration is in the range of 10–300 mM, the nonionic surfactant concentration is in the range of 0–3% w / v (or 0–1% w / v in some exemplary implementations), the pH of the final mixture may be in the range of 7.8–10 (or 8.2–9.5 in some exemplary implementations), and the antifoaming agent concentration is 0.005–0.5% (v / w).

[0224] The results shown in the following examples suggest that the size of debris particles is significantly influenced by the hemolytic reagent composition when processing blood samples for subsequent direct colony growth. However, the ability to achieve even smaller debris sizes by increasing the digestive capacity of the hemolytic reagent is limited by the requirement of microbial cell viability, particularly in the case of Gram-negative bacteria. This limitation is acceptable as long as the size and density of the debris do not interfere with the detection of microbial cells at the microcolony level. One example of a criterion in this regard is that, after diffusing the final cell suspension in solid growth medium, the surface of the solid growth medium should not be covered by lysis debris artifacts in an area fraction that exceeds 90%, preferably not exceeding 50%, and more preferably not exceeding 20%. We have found that this criterion can be met by treating whole blood samples with type 3 hemolytic reagent.

[0225] If the surface density of artifacts is not very high, it is possible to separate the background from the microcolonies by recording multiple images over a certain period of time using time-lapse imaging, or by removing the background, for example, by size thresholding as described above. To perform size selection, images of the gel surface can be taken from a limited area of ​​the surface before incubation or immediately after incubation. In one exemplary implementation, image analysis is performed to determine the average size R of the debris particles. back.av And the standard deviation sd of the debris particle size is measured, and the size threshold is R back.av And it can be determined based on sd. The size selection criterion is, for example, R>R 閾値 =R back.av +n * sd can be set, where n is an integer typically selected in the range of 1 to 6, and more typically n=3. In another embodiment, R back.av The SD can be predetermined (for example, provided with the cartridge or embedded in the device / instrument software) and made available during the colony analysis phase.

[0226] In some exemplary embodiments, the area on which the microbial suspension is seeded after lysing of host blood cells and separation from the whole blood sample is 50-200 mm² per 1 mL of initial whole blood sample to avoid the formation of a continuous layer of debris and to facilitate early detection of microbial cells. 2 It can be selected to be such that: In some exemplary implementations, the area of ​​the concentrated microbial suspension after lysis of host blood cells and separation from whole blood sample is 25-50 mm² per 1 mL of initial whole blood sample. 2 , or 50-100 mm per 1 mL of initial whole blood sample 2 , or 50-150 mm per 1 mL of initial whole blood sample 2 , or 50-300 mm per 1 mL of initial whole blood sample 2 It can be selected in such a way.

[0227] In some exemplary embodiments, conventional culture bottles can be used for sample collection, and the culture bottles can be optionally treated in the presence of an initial incubation to isolate microbial cells within the culture bottles, and a concentrated suspension of live microbial cells can be supplied to a solid growth medium for seeding. Blood culture bottles typically either contain resins for absorbing antimicrobial substances or rely on partial antimicrobial inactivation by commands containing sodium polyanethole sulfonate (SPS) and saponins, which are common additives for dissolving the bottles. Furthermore, the potential of antimicrobial substances decreases with approximately 5-fold dilution (ratio of culture medium to blood). Disadvantages of bottles as collection devices include: 1) the requirement of an approach to isolate cells from a large volume of about 40 mL, which is often cumbersome and difficult to automate; 2) problems with resin beads and medium particles during separation; and 3) blood coagulation, which affects the cleanliness of the final cell suspension.

[0228] Prior examples of hemolytic reagents and separation methods (centrifugation, filtration, microfluidics) have demonstrated various approaches for obtaining isolated suspensions of living microbial cells. These methods, and other methods well known in the art, can be adapted by those skilled in the art in combination with the selection of a suitable initial amount of sample to achieve a field density of debris after seeding on a solid medium that is sufficient to detect microcolonies according to a desired time detection window.

[0229] To achieve reliable microcolony detection within 2-3 hours, the inventors have found that microcolony detection typically occurs for microcolony diameters in the range of 20-70 μm, 20-60 μm, or 20-50 μm, resulting in desired post-seeding artifact surface coverage remaining in the range of less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, or less than 20%, and in some exemplary embodiments, 10-80%, 10-70%, 10-60%, 10-50%, 10-40%, 10-30%, or 15-30%. The inventors have also found that, in some cases, it may be useful to select dissolving reagents and separation methods such that the central debris particle size is, for example, less than 10 microns, less than 8 microns, or less than 6 microns, or for example, 1-10 microns, 1-8 microns, or 1-6 microns, in terms of the average lateral dimension measured in a plane parallel to the solid growth surface.

[0230] Parallel incubation of other detected microcolonies In some exemplary implementations, the detected second microcolony can be further incubated in parallel with nucleic acid amplification and / or sequencing based on microbial cells recovered from the detected first microcolony, and the cells derived from the detected second microcolony can then be used for downstream applications, such as performing microbial cell identification by MALDI, in a minimum number of cells (e.g., >10). 5The cells can be recovered after being measured to contain them. In some exemplary implementations, the detected third microcolony can be further incubated in parallel with nucleic acid amplification and / or sequencing based on microbial cells recovered from the detected first microcolony, and the cells derived from the detected third microcolony can then be recovered after being measured to contain a minimum number of cells for another downstream use, e.g., for performing antimicrobial susceptibility testing. Identification results, known species-specific breakpoints, and other clinical factors can be used when selecting antibiotics and dosages based on AST results.

[0231] As described above, in some exemplary embodiments, AST can be performed on microbial cells recovered from one or more colonies after at least presumptive identification or classification of the colony microbial cell class (presumptive identification may not be necessary, for example, when the colonies are recovered after reaching a size known to have the minimum number of cells across diverse microbial cell classes, and when a broad AST panel is used, for example, a panel broad enough to provide coverage for both sets of Gram-positive and Gram-negative bacteria). The recovered colonies (recovered using, for example, manual recovery, automated recovery, or a combination thereof) can then be suspended in a liquid to form a suspension (optionally diluted or concentrated), aliquoted, and contacted with various concentrations of antibiotics. Based on the identity of the microbial cells, the antibiotic (and optionally its concentration) can be selected. For example, aliquoted microbial cells can be contacted with three different concentrations of selected antibiotics, and the microbial cells can then be monitored to identify indicators of sensitivity and / or resistance. In other exemplary embodiments, further concentrations of antibiotics may be used.

[0232] Automated Workflow As described above, the methods disclosed herein can be implemented using manual, automated or semi-automated workflows. In some exemplary implementations, all of the method steps involving the separation of initial microbial cells, seeding of solid growth media, seeding of solid growth media and monitoring of microcolonies, recovery of microcolonies, preparation of microbial suspensions from the recovered microbial cells, optional lysis of the recovered microbial cells, and performance of nucleic acid amplification and / or sequencing based on the recovered microbial cells can be automated in a single instrument. In other exemplary embodiments, two or more instruments can be used, with each instrument performing one or more of the steps described above. For example, a first instrument, such as an automated cartridge-based centrifugation device disclosed in the specification of international patent application PCT / CA2015 / 050449, entitled "APPARATUS, SYSTEM AND METHOD FOR PERFORMING AUTOMATED CENTRIFUGAL SEPARATION", which is hereby incorporated by reference in its entirety, can perform the lysis of host cells and the separation of microbial cells. In some exemplary embodiments, the seeding step can be integrated, for example, with the separation step in a closed cartridge, as disclosed in the specification of international patent PCT / CA2019 / 051895.

[0233] Time and speed Unlike previous methods of colony growth and subsequent analysis, various exemplary embodiments of the present disclosure facilitate the rapid detection of infectious diseases by detecting the presence of microcolonies and rapid detection of nucleic acids by amplification and / or sequencing within hours after initially seeding a sample onto a solid growth medium, based on seeding of the solid growth medium in the absence of a preceding liquid culture step.

[0234] In some exemplary implementations, microcolonies can be recovered within 1.5 - 2.5 hours, 2 - 3 hours, 2 - 4 hours, and 2 - 5 hours from the initial processing of the sample for subsequent nucleic acid amplification.

[0235] In some exemplary implementations, nucleic acid amplification and / or sequencing may be initiated within 2-3 hours, 2-4 hours, 2-5 hours, and 2-6 hours from the initial processing of the sample.

[0236] In some exemplary implementations, nucleic acid amplification results can be obtained within 3–4 hours, 3–5 hours, and 3–6 hours from the initial processing of the sample.

[0237] In some exemplary implementations, microcolonies can be harvested for subsequent nucleic acid amplification within 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes from the initial microcolony detection.

[0238] In some exemplary embodiments, nucleic acid amplification and / or sequencing based on microbial cells recovered from microcolonies may be initiated within 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes from the recovery of the microbial cells.

[0239] In some exemplary embodiments, nucleic acid amplification and / or sequencing based on microbial cells recovered from microcolonies may be initiated within 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes after lysing the microbial cells recovered from the microcolonies.

[0240] Lysis of microbial cells As described above, after lysing the recovered microbial cells, nucleic acid amplification reactions and / or sequencing are performed. As detailed below, the inventors have discovered that nucleic acids can be detected without problems from the recovered microcolonies using a variety of lysis methods. Non-limiting exemplary methods of lysis include bead crushing, ultrasonic dissolution, thermal dissolution, and electrolysis.

[0241] Contrary to conventional expectations in the art that thermal lysis is usually insufficient for lysing Gram-positive microbial cells, the inventors have discovered the surprising result that thermal lysis can be an effective method for lysing a wide range of Gram-negative and Gram-positive pathogens.

[0242] In other exemplary embodiments, dissolution may be carried out electrically, for example, according to a method disclosed in the international patent application PCT / CA2012 / 000698, entitled “METHODS AND DEVICES FOR ELECTRICAL SAMPLE PREPARATION,” which is incorporated herein by reference in its entirety. For example, a suspension of microbial cells obtained by suspending recovered microbial cells in a liquid (having an ionic strength of 0.1 mM to 10 mM) may be electrically processed in a fluid channel by applying a bipolar voltage pulse between an upper electrode and a lower electrode such that the electric field generated across the thickness of the fluid channel is approximately 2 kV / cm to 30 kV / cm, and the liquid is heated at a heating rate of approximately 250 °C / sec.

[0243] To demonstrate this efficiency of lysis from cells recovered from microcolonies, and as described in the examples below, we performed thermal lysis followed by real-time reverse transcriptase loop-mediated isothermal amplification (rRT-LAMP). rRT-LAMP is a molecular diagnostic technique that selectively detects RNA templates under isothermal conditions, typically 60–65°C. For this experiment, we designed six different primer sets for the detection of bacterial cells / species with high specificity to either the species or genus level (as shown in Figure 9A) by transcription, and for the amplification of ribosomal RNA released by thermal lysis. As known from prior art, thermal lysis, while simple and easy to use, is highly inefficient for releasing ribosomes from Gram-positive bacteria.

[0244] The experiment was conducted according to the method of Example 9, and the results are shown in Figure 9A. In this case, assay performance is measured by "Time to Positive (TTP)". A short TTP indicates that a robust assay, as time and TTP > 15 minutes, is judged to be a false-positive detection resulting from nonspecific amplification of contamination. In contrast to Gram-negative bacteria, the stronger the cell wall of mature Gram-positive bacteria, the less ribosomes (source of rRNA templates) are released, and therefore poor performance for Gram-positive bacteria was expected. This unexpected observation highlights one advantage of using cells recovered from microcolonies for molecular assays, considering that the implementation of thermal lysis in the cartridge is easier. Even more surprising results are shown in Figure 9B. Here, the performance of the RT-LAMP assay is compared for two cases: 1) when the cell suspension is subjected to thermal lysis, and 2) when cell lysis is not performed before RT-LAMP. As observed, the assay performance is similar except for the case of Klebsiella pneumoniae. Interestingly, even in this case, acceptable performance was obtained in the assay using cell suspensions obtained from 4-hour-old microcolonies.

[0245] The advantage of recovering cells from microcolonies in terms of ease of cell lysis was further demonstrated by PCR detection of rRNA genes using genomic DNA (gDNA). For this purpose, two primer sets were designed for the detection of highly specific E. coli or Staphylococcus aureus bacterial species. Experiments were performed according to the method of Example 10. In our experience, assay performance is considered acceptable when the CT value is less than 35 cycles. Naturally, accidental contamination from environmental templates may increase false positive values. However, these accidental cases have CT values ​​greater than 32 cycles, and smaller CT values ​​correspond to robust detection.

[0246] Figure 10A shows the CT values ​​corresponding to PCR detection of Escherichia coli or Staphylococcus aureus bacterial species obtained from 4 or 6-hour-old microcolonies. The assay performance is excellent in both cases using thermal lysis and electrolysis.

[0247] In another experiment, we investigated the possibility of performing a robust PCR assay with cell suspensions obtained from microcolonies in the absence of a cell lysis step. The results, shown in Figure 10B, surprisingly show that the performance is similar to that achieved using thermal lysis. These observations highlight the advantage of obtaining cells from microcolonies, suggesting that in some cases, cell lysis can be avoided or, for example, limited by thermal or electrolysis, thereby enabling suitable lysis even in the case of Gram-positive bacterial cells.

[0248] In contrast to glass bead lysis, lysis methods such as thermal and electrolysis may be beneficial in that they prevent the release of integrated gDNA upon lysis. Therefore, thermal and / or electrolysis may be useful for sequencing applications where nucleic acid fragments are no longer desired. On the other hand, glass bead lysis can be useful as a lysis method for bacterial identification by PCR amplification of specific regions of gDNA, as it shears gDNA and thus promotes the amplification of multiple copies of the gene (about 10 in the case of rRNA genes). However, this latter aspect may not be very important when obtaining cells from microcolonies due to the large number of cells.

[0249] Examples of solid growth media types It will be understood that the growth and detection of microcolonies can be facilitated by using a variety of solid growth media. Solid growth media provide microbial cells along with a suitable source of growth medium and are suitable for supporting colony formation during incubation. Non-limiting examples of solid growth media include conventional agar, gelatin, guar gum, and xanthan gum, which have suitable growth nutrients. In some exemplary implementations, solid growth media may be chromogenic depending on the type of microbial cell. In some embodiments, a chromogenic substrate or fluorescent substrate may be added to the agar medium to identify microorganisms by specific or nonspecific staining of colonies.

[0250] The solid growth medium may be in a dry or partially dry format so that it is absorbed (at least partially) from the liquid component of the cell suspension upon contact.

[0251] In some exemplary embodiments where the solid growth medium contains agar, the agar concentration can be between 1.5% and 2.0%, which allows for a substantially harder surface than gels formed from lower concentrations of agar. We have found that such a hard surface is less susceptible to surface damage during microcolony retrieval. This embodiment is advantageous when it is desired to maintain the growth of microcolonies for subsequent retrieval episodes. Furthermore, the likelihood of gel fragments transferring to the resulting cell suspension is substantially reduced. Such gel fragments, if not removed by methods such as filtration, can degrade the performance of downstream assays. As taught in Example 3, in one embodiment, the gel composition is optimized for the growth of preferred organisms.

[0252] Rapid Gram staining of microbial cells recovered from microcolonies While previous exemplary embodiments have disclosed methods for using microbial cells recovered from microcolonies for rapid nucleic acid amplification or sequencing, we have also discovered that microbial cells recovered from microcolonies can be used directly for Gram staining. Thus, such microcolony-based Gram staining methods enable both (ii) rapid detection of infectious diseases by rapid microcolony detection within hours of the start of sample processing, and (ii) measurement of the Gram status of the detected microcolonies. In some exemplary implementations, rapid microcolony-based Gram staining can be performed on one detected microcolony immediately after or after microcolony detection, allowing another microcolony to be processed separately for AST, and the rapid microcolony-based Gram staining results to select an appropriate subset of antimicrobial agents for AST.

[0253] The following Example 1A shows experimental results demonstrating the ease and clarity of rapid Gram staining based on microcolony samples.

[0254] Improved method and hemolytic reagent for whole blood sample processing As mentioned above, many attempts have been made to facilitate the isolation of microbial cells from whole blood in order to enable subsequent processing that avoids the long delays associated with liquid blood cultures.

[0255] For example, the Isolator®, developed by Dorn in the 1970s and refined in the 1980s, is a sample collection and transport container that holds a hemolytic reagent containing saponins and sodium polyanethole sulfonate (SPS). After collecting whole blood samples in the container and mixing them with the hemolytic reagent, the container is transferred to the laboratory, subsequently centrifuged, and plated with solid growth medium to obtain concentrated microbial cells. Approximately 24–48 hours after seeding, colonies are usually detectable in the growth medium.

[0256] Although Isolator® does not require liquid blood cultures, it suffers from the same long detection time associated with liquid blood cultures, due to a delay before macroscopic colony detection. Recognizing that this drawback of Isolator® is associated with insufficient digestion and removal of residual blood debris, the inventors embarked on developing improved hemolytic reagent formulations and methods to facilitate the detection of microbial cells within minutes or hours of whole blood isolation. The inventors began pursuing improvements to Dorn's method based on hemolytic reagent compositions containing saponins and SPS.

[0257] As stated above, International Patent Specification PCT / CA2019 / 050716, titled "METHODS AND COMPOSITIONS FOR THE SELECTIVE LYSIS OF BLOOD CELLS AND SEPARATION OF MICROBIAL CELLS," discloses that improved hemolytic reagents and microbial isolation methods further enhance blood cell lysis and digestion by selectively combining the hemolytic advantages of saponins, the microbial protective and anticoagulant advantages of SPS, and the improved blood debris decomposition capacity and low viscosity provided by the alkaline environment with an increased saponin concentration compared to that of Isolator®.

[0258] Figure 18 summarizes the hemolysis workflow described in this patent application. The hemolysis reagent containing saponins and SPS is stored separately from the alkaline buffer, as shown in 200. Before adding the whole blood sample, the hemolysis reagent is pre-mixed with the alkaline buffer to form the alkaline hemolysis reagent containing saponins and SPS, as shown in 210. Then, as shown in step 220, the whole blood sample is mixed with the alkaline hemolysis reagent to form a mixture. Then, in step 230, microbial cells are separated from the mixture using a separation process such as centrifugation, filtration, or microfluidic separation to obtain a cell suspension.

[0259] The hemolysis formulation and method shown in Figure 18 demonstrated that it enables improved digestion of residual blood debris and facilitates direct molecular processing of isolated microbial cells. Furthermore, as disclosed in PCT patent application PCT / CA2019 / 051895, entitled "SYSTEMS AND METHODS FOR MICROCOLONY GROWTH AND MICROBIAL CELL CHARACTERIZATION," the whole blood lysis and isolation method shown in Figure 18 demonstrated that microcolony detection is possible within a few hours of inoculation for a variety of bacterial and fungal species. This is in stark contrast to cases where several days are required to detect colonies after inoculation of concentrates obtained from Isolator®.

[0260] The alkaline saponin and SPS-based hemolytic reagents disclosed in PCT / CA2019 / 050716 were found to be particularly susceptible to the effects of processing whole blood samples in the range of 1–3 mL. However, the inventors have found that processing larger volumes of whole blood samples can increase the amount of residual hemolytic debris, which may lead to performance limitations in some downstream applications. For example, the inventors have found that residual blood debris resulting from larger sample volumes may impair the ability to rapidly detect isolated microbial cells and / or rapidly detect microcolonies that have grown from isolated microbial cell suspensions. It has also been found that, for larger volumes of whole blood samples, residual blood debris can lead to the enclosure of microbial cells during the separation process, resulting in losses in recovery, which can be a significant consequence for low-titer whole blood samples known to exhibit microbial cell counts in the range of 0.1–10 CFU / mL.

[0261] The inventors aimed to develop an improved workflow that would increase the efficiency of digesting and removing residual blood debris and facilitate the processing of blood samples having sample volumes in the range of 5-10 mL (or a larger volume range, e.g., 5-15 mL or 5-20 mL), and worked to improve the hemolytic reagent and microbial cell isolation method taught in PCT / CA2019 / 050716. Since improved digestion and removal of blood debris can be achieved by improving the method shown in Figure 18, the hypothesis was made that, instead of pre-mixing the saponin and SPS-containing hemolytic reagent with the alkaline buffer before adding the whole blood sample, the saponin and SPS-containing hemolytic reagent should first be brought into contact with and mixed with the whole blood sample before adding the alkaline buffer.

[0262] This modified method is shown in Figure 19, which differs from the method in Figure 18 in that it begins by mixing the whole blood sample with a hemolytic reagent containing ponin and SPS, as shown in step 300, before contact with the alkaline buffer. After the formation of the first mixture, as shown in step 320, the first mixture is mixed with the alkaline buffer to form the second mixture. An alkaline buffer is selected that has a pH and ionic strength such that the second mixture is alkaline.

[0263] As shown in step 330, a separation process is then used to separate the microbial cells from the second mixture and provide a purified microbial cell suspension. Non-limiting examples of preferred separation methods include centrifugation comprising one or more optional washing and centrifugation steps, filtration, microfluidic separation, and immunomagnetic separation. An exemplary automated system for carrying out the separation and concentration of microbial cells is described in international patent application PCT / CA2015 / 050449.

[0264] Next, the isolated microbial cells, in the form of a microbial suspension as shown in Figure 19, can be processed according to various growth and / or analytical steps, examples of which will be described later.

[0265] While not intended to be constrained by theory, the inventors hypothesized that a modified approach, as shown in Figure 19, in which the hemolytic reagent and alkaline buffer are added sequentially instead of being pre-mixed before contact with the whole blood sample, and the whole blood sample is first contacted with the hemolytic reagent before the addition of the alkaline buffer, would promote more efficient lysis and degradation of blood cells during the initial contact phase with the hemolytic reagent. The experiments to test this hypothesis are described below.

[0266] Furthermore, we hypothesized that if a time delay occurs before the addition of the alkaline buffer and the separation of microbial cells in an exemplary workflow where the first mixture is initially formed when the whole blood sample is collected from the patient or immediately thereafter, and then transferred to another location (such as a laboratory or a central sample processing facility), the higher viscosity of the first mixture compared to the low viscosity that occurs after the addition of the alkaline buffer would provide at least some protection from antimicrobial agents that may be present in the whole blood sample, thereby protecting the viability of microbial cells during sample transport.

[0267] As shown in Figure 19, the improved continuous workflow may include a time delay between (i) mixing the whole blood sample with the hemolytic reagent to form a first mixture and (ii) adding the alkaline buffer. This time delay may occur, at least in part, while the first mixture is being moved from the blood collection location to the sample processing location.

[0268] In some exemplary implementations, the hemolytic reagent may be stored in a partially vented container, such as a partially vented tube, at a pressure low enough to facilitate the collection of a desired volume of whole blood sample. Mixing the whole blood sample with the hemolytic reagent yields a first mixture in which at least partially the blood cells are lysed and the blood cell debris is digested. The container can be moved to a separate processing location, during which at least partially the delay 310 shown in Figure 19 may occur.

[0269] In some exemplary implementations, an alkaline buffer can be stored in a chamber within a fluid cartridge configured to operate for the isolation of microbial cells. The first mixture can be added to the fluid cartridge and mixed with the alkaline buffer by a manual, automated, or semi-automated method. Non-limiting examples of automated equipment and fluid cartridges using centrifugation for microbial cell isolation are disclosed in international patent application PCT / CA2015 / 050449 entitled "APPARATUS, SYSTEM AND METHOD FOR PERFORMING AUTOMATED CENTRIFUGAL SEPARATION".

[0270] In some exemplary implementations, a fluid cartridge can be provided that contains both a hemolytic reagent and an alkaline buffer, the hemolytic reagent being contained in a first chamber within the fluid cartridge, and the alkaline buffer being stored in a second fluid chamber within the fluid cartridge, the first and second chambers not initially in fluid communication via one or more valves, provided, for example, in a (passively) closed state. The first chamber containing the hemolytic reagent may be provided in a partially ventilated state by a pierceable and resealable top (e.g., a stopper) made of rubber, elastomer, or other suitable material, and the first chamber may have an initial pressure low enough to facilitate the passive collection of a desired volume of whole blood sample and form a first mixture. The cartridge can then be moved to a position where an automated device is located, and one or more valves close during the movement to prevent fluid contact of the first mixture with the alkaline buffer, and a delay 310 in Figure 19 occurs during the movement to facilitate hemocellular lysis and digestion of blood debris. Next, the fluid cartridge is processed by automated equipment, one or more valves are opened, and the first mixture is mixed with an alkaline buffer according to the method in Figure 19 to obtain a second mixture. One or more additional automated steps can then be performed to separate the microbial cells from the second mixture (e.g., by centrifugation, filtration, microfluidic separation, and immunomagnetic separation).

[0271] In step 310 of Figure 19, the inventors conducted experiments to study the effects of the resulting blood lysis efficiency and the time delay in the digestion of blood debris (the delay between mixing the whole blood sample with a blood lysis reagent containing saponin and SPS to form a first mixture, and subsequently mixing the first mixture with an alkaline buffer to form a second mixture).

[0272] A large 7 mL sample volume of whole blood was collected in a vent tube containing 2 mL of hemolytic reagent containing saponin and SPS. The hemolytic reagent (before sample addition) had the following composition: Tergitol® L-62 (25 mM or 50 mM); 0.20% w / w, methyl-β-cyclodextrin (MBCD); 6.25 mM, SPS; 4.25% w / w, triterpenoid saponin; 5.00% w / w, acetate buffer: 150 mM. Following the method shown in Figure 19, the hemolytic reagent was mixed with the whole blood sample by inverting the tube several times to form the first mixture. Subsequently, 20 μL aliquots were collected every 10 minutes for microscopic imaging and viscosity measurement. Alkaline buffer (0.15 M Tris(tris(hydroxymethyl)aminomethane) buffer) was added after 40 minutes. Microscopic imaging of the mixture was performed according to Wright's staining method, as described in Example 16.

[0273] Figure 20 shows the obtained microscopic images. This reveals that the lysis of blood cells and digestion of blood debris by the hemolytic reagent are time-dependent. At t=0, the smear on the microscope slide was too thick to reveal the internal morphology, and the image lacks characteristic features. At 10 minutes, most of the red blood cells had lysed, and only platelets and leukocytes were observed. After 20 minutes, platelets and leukocytes were no longer distinguishable, and only elongated filamentous structures were observable. After 40 minutes, the filamentous structures were no longer observable.

[0274] The time dependence of the viscosity of the first mixture after its formation and the decrease in viscosity after the addition of the alkaline buffer were estimated by measuring the distance traveled by a 10 μL droplet on a plastic sheet and comparing it with the distance traveled by a droplet of a body fluid solution (body fluid viscosity reference) on the same sheet. As can be seen in Figure 20, before the addition of the alkaline buffer, the viscosity was observed to increase over the first 20 minutes, and then slowly decrease to a value of approximately 90 cP after 40 minutes.

[0275] After 40 minutes, an alkaline buffer was mixed with the first mixture to obtain a second mixture with a basic pH. The addition of the alkaline buffer increased the pH of the mixture to 8.5 and significantly reduced its viscosity from 90 cP to 7 cP. This viscosity reduction, beneficial for facilitating efficient microbial cell isolation and debris removal by filtration, centrifugation, or other separation methods (e.g., microfluidic separation), was observed to occur independently of the time delay between contacting the whole blood sample with the hemolytic reagent to form the first mixture and then contacting the first mixture with the alkaline buffer to form the second mixture.

[0276] Although Figure 20 shows that no filamentous structures are present in the microscopic image taken after the addition of the alkaline buffer, the inventors have discovered that such filamentous structures persist even after the addition of the alkaline buffer if the time between the formation of the first mixture (mixing of the hemolytic reagent with the whole blood sample) and the addition of the alkaline buffer is insufficient. If the time before the addition of the alkaline buffer is insufficient, such filamentous structures may be observed floating inside the liquid.

[0277] While not intended to be theoretically bound, it appears that such residual filamentous structures, if present due to incomplete degradation, can trap microbial cells. Therefore, these residual filamentous structures can obstruct the separation process by clogging and reduce microbial cell recovery due to the loss of microbial cells during separation (e.g., centrifugation or filtration). Such loss of microbial cells (loss of recovery) can be particularly detrimental when the microbial cell concentration in the original sample is in the range of 0.1–10 CFU / mL.

[0278] Therefore, in some exemplary embodiments, a delay is provided at 310 as shown in Figure 19 between the time for the formation of the first mixture, i.e., the time when hemolysis begins (by mixing the whole blood sample with the hemolytic reagent), and the time for the first mixture to be brought into contact with (mixed with) an alkaline buffer to obtain the second mixture, i.e., the time when basicization begins. In some exemplary implementations, the delay between forming the first mixture and bringing the first mixture into contact with an alkaline buffer to form the second mixture is 15 to 30 minutes, or 15 minutes to 1 hour, 15 minutes to 2 hours, or 15 minutes to 3 hours, or 15 minutes to 4 hours, or 15 minutes to 5 hours, or 15 minutes to 6 hours, or 15 minutes to 8 hours, or 15 minutes to 10 hours, or 15 minutes to 12 hours, or 15 minutes to 14 hours, or 15 minutes to 16 hours, or 15 minutes to 20 hours, or 15 minutes to 22 hours, or 15 minutes to 24 hours. In some exemplary implementations, the delay between the formation of the first mixture and its contact with the alkaline buffer is 30 minutes to 1 hour, 30 minutes to 2 hours, or 30 minutes to 3 hours, or 30 minutes to 4 hours, or 30 minutes to 5 hours, or 30 minutes to 6 hours, or 30 minutes to 8 hours, or 30 minutes to 10 hours, or 30 minutes to 12 hours, or 30 minutes to 14 hours, or 30 minutes to 16 hours, or 30 minutes to 20 hours, or 30 minutes to 22 hours, or 30 minutes to 24 hours. In some exemplary implementations, the delay between the formation of the first mixture and its contact with the alkaline buffer is 340 minutes to 1 hour, 40 minutes to 2 hours, or 40 minutes to 3 hours, or 40 minutes to 4 hours, or 40 minutes to 5 hours, or 40 minutes to 6 hours, or 40 minutes to 8 hours, or 40 minutes to 10 hours, or 40 minutes to 12 hours, or 40 minutes to 14 hours, or 40 minutes to 16 hours, or 40 minutes to 20 hours, or 40 minutes to 22 hours, or 40 minutes to 24 hours.

[0279] In some exemplary implementations, the delay between the formation of the first mixture and its contact with the alkaline buffer is sufficiently long, and after the delay, but before the addition of the alkaline buffer, the first mixture lacks red and white blood cells under microscopic evaluation.

[0280] In some exemplary implementations, the delay between the formation of the first mixture and its contact with the alkaline buffer is sufficiently long, and after the delay, but before the addition of the alkaline buffer, the first mixture lacks filamentous structures under microscopic evaluation.

[0281] In some exemplary implementations, the delay between the formation of the first mixture and its contact with the alkaline buffer is sufficiently long, and the first mixture is (i) contacted with the alkaline buffer to separate the microbial cells from the second mixture to obtain a suspension of microbial cells, and after seeding the suspension onto a solid growth medium, the area coverage of residual blood debris on the solid growth medium is 5%-50%, 5%-40%, 5%-30%, or 5%-20%.

[0282] The inventors have found that by including a delay between the formation of the first mixture and its contact with the alkaline buffer, it may be possible to process larger volumes of whole blood without significantly increasing residual blood debris compared to a workflow that involves pre-mixing the hemolytic reagent with the alkaline buffer before adding the whole blood sample.

[0283] Volume of whole blood V B However, the inventors have found that this is an important parameter governing the purity of the microbial cell suspension obtained after separation. Specifically, it has been found that the larger the blood volume, such as 5 mL to 10 mL, the lower the microbial load in a typical clinical whole blood sample, making it more susceptible to and sensitive to the harmful effects of insufficient recovery. Therefore, some aspects of this disclosure relate to methods suitable for processing such large blood volumes.

[0284] To illustrate the beneficial effect of time delay on the ability to process large volumes of whole blood samples without generating significant blood-soluble debris, various volumes of whole blood (V) were used in each case. B ), blood lysis reagent (V L ) and the total volume of blood that has been dissolved and made basic (V L+BAn experiment was conducted to investigate three cases using ), and in only one case there was a time delay T between hemolysis and basicization. b In all cases, the hemolytic reagent had the following composition: Tergitol® L-62; 0.20% w / w, MBCD; 6.25 mM, SPS; 4.25% w / w, saponin; 5.00% w / w, acetate buffer: 150 mM. Basicization was achieved by adding Tris buffer so that the ionic strength of the final solution was either 20 mM or 60 mM and the pH of the final solution was either 8 or 8.8.

[0285] The volume and time for the three cases were as follows: Case A: Following the method shown in Figure 18, the hemolytic reagent was pre-mixed with an alkaline buffer to form an alkaline hemolytic mixture. The alkaline mixture was then mixed with whole blood collected in a standard SPS tube; V B = 5 mL, V L =3mL, V L+B = 8 mL. Case B: Following the method shown in Figure 18, the hemolytic reagent was pre-mixed with an alkaline buffer to form an alkaline hemolytic mixture. The alkaline mixture was then mixed with whole blood collected in a standard SPS tube; V B =2.5mL, V L =3.5mL, V L+B = 6 mL. Case C: Following the method described in Figure 19, whole blood is collected in a vent tube containing a hemolytic reagent, and then T b = Mixed with alkaline buffer after a 30-minute delay; V B =6mL, V L =2mL, V L+B = 8 mL.

[0286] For each case, the basicized mixture was separated by centrifugation according to the method of Example 19, washed, and the separated microbial cells were resuspended to form a microbial cell suspension. 100-120 μL of the obtained cell suspension was grown on an agar plate and observed under a microscope after 30 minutes. The average debris particle size and surface coverage were measured. These are shown in Figure 21.

[0287] Using the premixing method shown in Figure 18, Case A, with a large sample volume of 6 mL, was still affected by the complete coverage of the solid growth medium, preventing colony detection during the early microcolony stage for both final alkaline pH values ​​of 8 and 8.8.

[0288] The results for Case B, which also used the premixing method shown in Figure 18, were inferior to those for Case A. Although the volume of whole blood used was half that of Case A, only 3 mL of whole blood was processed in Case B. In fact, both experimental implementations of Case B (with final pH values ​​of 8 and 8.8) resulted in significantly larger central blood lysis debris particle sizes and even larger area fractions.

[0289] In stark contrast, in Case C, using the improved continuous method shown in Figure 19, where there was a 30-minute time delay between hemolysis and basicization, a low average debris size (less than 4 μm) and a low-area surface coverage fraction of less than 30% were achieved. Even with a large whole blood sample volume of 6 mm, microcolony detection was possible without significant performance loss in terms of time to positivity.

[0290] The blood lysis reagents described herein use saponins as solvents, and in some embodiments, specific types, concentration ranges, and / or purities of saponins may be used. Saponins are naturally occurring surfactant glycosides produced by plants, lower marine animals, and some bacteria. Saponins exhibit surfactant activity and consist of a carbohydrate moiety bound to a hydrophobic aglycone (sapogenin). Based on the aglycone structure from which they originate, saponins are broadly classified as triterpenoids, steroids, or steroidal glycoalkaloids. The variability of the aglycone structure, the properties of the side chains, and the binding sites of these sites on the aglycone are the main causes of structural diversity in various saponins.

[0291] The membrane activity of both triterpenoids and steroid saponins is known to be associated with their ability to interact with membrane-bound cholesterol. Upon binding to cholesterol, saponins induce changes in membrane structure, thereby increasing permeability. At concentrations higher than their critical micelle concentration (CMC), saponins form aggregates, which are thought to bind to cholesterol within the red blood cell membrane, creating a large, stable core that ultimately leads to cytolysis. This selectivity for cholesterol contained within the cell membrane allows saponins to lyse red blood cells while leaving bacterial cells intact and viable in the same medium.

[0292] Triterpenoid saponins can be found in legumes such as alfalfa, chickpeas, broad beans, soybeans, lentils, kidney beans, peanuts, sunflower seeds, ginseng root, horse chestnut, tea leaves, licorice root, quillaja bark, spinach leaves, quinoa seeds, tea leaves, sugar beets, and other garlic species, while steroidal saponins are found in yucca, tomato seeds, ginseng root, yam, eggplant, fenugreek seeds, asparagus, and chili peppers (Oleszek & Oleszek, 2020). The main source of industrial triterpenoid saponins is the bark of quillaja saponaria, a tree native to the Andes region. Saponins represent 20–25% of the extractable material derived from their source (Barr, et al., 1998, Ad Drug Deliv Rev, 32:247-271).

[0293] Saponin solutions are often purified by filtration using filtration devices with various types of membranes after dissolution. The effectiveness of filtration can vary depending on the source of the saponins, due to differences in the degree of refinement used by various commercial vendors. For example, commercial extracts of unpurified quillaya saponins typically contain about 20% by weight of triterpenoid saponins, while higher-purity semi-purified extracts can contain 75-80% by weight of triterpenoid saponins (San Martin et al., 2000, J.Sci. Food Agric., 80:2063-2068).

[0294] Quillaja saponins are generally weakly acidic. For example, when dissolved in water at a concentration of 20% by weight, quillaja saponins can exhibit a pH of 4-5 depending on their purity. When the pH is greater than 8, the main quillaja saponin compounds, QS-7, QS-17, QS-18, and QS-21, undergo alkaline hydrolysis to produce the "deacetylated saponins" DS-1 and DS-21. These deacetylated saponins exhibit approximately 10 times lower hemolytic activity than their parent saponins (DJPillion, JAAmsden, CRKensil, J. Recchia “Structure-function relationship among Quillaja saponins serving as excipients for nasal and ocular delivery of insulin.” J of Pharmaceutical Sciences, Vol 85, No. 5 1996, pages 518-524). Thus, as the pH value increases, the critical micelle concentration of the saponin mixture also increases tenfold, from 200 mg / L at pH 6.5 to 2000 mg / L at pH 10 (WJ Chen, LC Hsiao, K KY Chen “Metal desorption from copper(II) / nickel(II)-spiked kaolin as a soil component using plant-derived saponin biosurfactant.” Process Biochemistry, Vol.43, No 5 2008, pages 488-498).

[0295] As mentioned above, the saponin material used to prepare hemolytic reagents can be obtained from the crushed endobark or woody aqueous extract of Quillaja saponaria Molina. This early extract consists of less than 20% w / w triterpene saponins, but in solution, it is unstable due to the precipitation of protein-polyphenol complexes. To remove some of the polyphenols and proteins, the extract can be processed to increase its purity and concentration. For example, the saponin extract can be treated with albumin, gelatin, vegetable protein, or a superabsorbent polymer such as polyvinylpolypyrrolidone and bentonite, and then filtered through diatomaceous earth to increase the saponin content to 20-26% by weight. At this stage, the hemolytic activity against sheep's blood is usually about 150-400 μg / mL.

[0296] To further purify the saponin extract, additional purification steps can be used, such as purification by diafiltration and ultrafiltration or tangential flow filtration using a membrane with a molecular weight cutoff of 10–75 kilodaltons. After such additional purification steps and spray drying, the dry saponin reagent can be increased to over 75% w / w and often up to 85% w / w and beyond (as measured by ULC, for example). Such additional purification steps also reduce the concentrations of impurities such as polyphenols, polysaccharides, and calcium salts.

[0297] The purified saponin material used to prepare the hemolytic reagent may have a purity of 75-96% by weight (%w / w) triterpene saponin, or 80-96% by weight (%w / w) triterpene saponin, or 85-96% by weight (%w / w) triterpene saponin, or 75-90% by weight (%w / w) triterpene saponin, or 80-90% by weight (%w / w) triterpene saponin, or 85-90% by weight (%w / w) triterpene saponin, or 75-85% by weight (%w / w) triterpene saponin, or 80-85% by weight (%w / w) triterpene saponin. The purified saponin material used to prepare the hemolytic reagent may contain impurities such as polyphenols and tannins (0.5-2.0% w / w), polysaccharides (1-5% w / w), plant proteins (2-5% w / w), and calcium salts such as calcium oxalate (0.2-1.0% w / w). When the purified saponin material containing 85% triterpene saponins was measured by a quantitative dose-response assay, it was found to have a hemolytic activity (HC50) of 55-88 μg / mL against a 4% suspension of purified sheep erythrocytes in 0.9% w / v sodium chloride in phosphate buffer (the dose-response measurement for purified sheep erythrocytes is used as a proxy for the activity against human erythrocytes).

[0298] In some exemplary implementations, the hemolytic reagent used to process a whole blood sample may contain an amount of saponin such that, after mixing the hemolytic reagent with the whole blood sample to form a first mixture, the concentration of saponin in the first mixture is 0.75–60 mg / mL, or 3–60 mg / mL, or 5–60 mg / mL, or 10–60 mg / mL, or 15–60 mg / mL, or 20–60 mg / mL, or 30–60 mg / mL, or 30–50 mg / mL, or 20–40 mg / mL, or 10–30 mg / mL, or 5–30 mg / mL, or 3–25 mg / mL, or 0.75–25 mg / mL.

[0299] In some exemplary implementations, the hemolytic reagent used to process a whole blood sample may contain an amount of SPS such that, after mixing the hemolytic reagent with the whole blood sample to form a first mixture, the concentration of SPS in the first mixture is 0.35–50 mg / mL, or 1–50 mg / mL, or 2–50 mg / mL, or 5–50 mg / mL, or 10–50 mg / mL, or 20–50 mg / mL, or 20–30 mg / mL, or 15–30 mg / mL, or 10–30 mg / mL, or 5–30 mg / mL, or 2–30 mg / mL, or 1–30 mg / mL, or 0.35–30 mg / mL.

[0300] Hemolytic reagents can be prepared to have a pH of 3.5-8, or 3.5-7, or 3.5-6.95, or 3.5-6.5, or 3.5-5.5, or 4-8, or 4-7, or 4-6.95, or 4-6.5, or 4-5.5, or 4.5-8, or 4.5-7, or 4.5-6.95, or 4.5-6.5, or 4.5-5.5. Examples of buffers for preparing hemolytic reagents include, but are not limited to, acetate, citrate, ascorbate, benzoate, or malate buffers.

[0301] In some exemplary implementations, the alkaline buffer may have pH values ​​of 7.3–10.5, 7.5–9.5, 7.6–9, and 8–9. Examples of alkaline buffers include, but are not limited to, triethanolamine, N-[tris(hydroxymethyl)methyl]glycine (tricine), 2-amino-2-(hydroxymethyl)-1,3-propanediol (Tris base), N,N-bis(2-hydroxyethyl)glycine (bicine), N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid (AMPSO), 2-(cyclohexylamino)ethanesulfonic acid (CHES), 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPSO), 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS), and sodium carbonate. The ionic strength of the alkaline buffer can be selected based on the composition of the alkaline buffer and its downstream application (e.g., whether the downstream application requires or benefits from maintaining microbial cell viability). Exemplary ranges of ionic strength for the alkaline buffer in the final solution (blood and hemolytic mixture) include, but are not limited to, 10–100 mM, 20–60 mM, or 25–40 mM.

[0302] The hemolytic reagent may have a volume of 0.5–2.0 mL, or 0.5–2.5 mL, or 0.5–3.0 mL, or 0.5–4.0 mL, or 2.0–4.0 mL, or 2.0–3.0 mL. In some exemplary embodiments, a hemolytic reagent can be prepared having a volume that falls within one or more of the aforementioned concentration ranges for saponin and SPS, so that when mixed with 3 mL of whole blood sample, at least one of these ranges is obtained in the first mixture. The hemolytic reagent may have a volume that falls within one or more of the aforementioned concentration ranges for saponin and SPS, so that when mixed with 5 mL of whole blood sample, at least one of these ranges is obtained in the first mixture. The hemolytic reagent may have a volume that falls within one or more of the aforementioned concentration ranges, so that when mixed with 10 mL of whole blood sample, at least one of these ranges is obtained in the first mixture.

[0303] The inventors have found that the ability to produce a first mixture that is substantially free of precipitate or contains a sufficiently small amount of precipitate, thereby enabling the desired performance of downstream detection sensitivity and / or assay performance, may depend on achieving a sufficient level of purity (sufficiently low concentration) of one or more reagents in the hemolytic reagent.

[0304] For example, after mixing the first mixture, a large amount of polyphenols can potentially co-precipitate with other blood proteins such as hemoglobin. Therefore, in some exemplary implementations, the hemolytic reagent used to process a whole blood sample may contain an amount of polyphenols such that, after mixing the hemolytic reagent with the whole blood sample to form the first mixture, the polyphenol concentration is 0.02–1.3% w / w, or 0.04–0.4% w / w, or 0.1–0.3% w / w.

[0305] The inventors have also discovered that some impurities in blood lysis reagents can lead to instability during storage before use by forming colloidal complexes with, for example, polyphenols, polysaccharides, and proteins. In fact, the inventors have found that the weight percentage of calcium in the hemolytic reagent (measured, for example, by atomic absorption spectroscopy) relative to the dry weight of saponin in several hemolytic formulations is 0.1% to 1%, or 0.1% to 0.75%, or 0.1% to 0.6%, or 0.1% to 0.5%, or 0.1% to 0.4%, or 0.05% to 1%, or 0.05% to 0.75%, or 0.05% to 0.6%, or 0.05% to 0.5%, or 0.05% to 0.4%, or 0.25% to 1%, or 0.25% to 0.75%, or 0.25% to 0.6%, or 0.25% to 0.5%, or 0.25% to 0.4%. The inventors have discovered that, in some cases, if the calcium content by weight exceeds 0.5% relative to the dry weight of the saponin, turbidity may be observed in blood lysis reagents within one week of storage at room temperature.

[0306] It has also been found that large amounts of calcium and polyphenols, when present together, can precipitate from the solution as calcium phenolates, particularly at pH levels greater than 9. Therefore, especially when the alkaline buffer is configured to yield a pH of 9 or higher in the second mixture and / or when the alkaline buffer uses a carbonate / bicarbonate buffer system (which can result in calcium carbonate precipitation), it may be beneficial to ensure that the total calcium content in the hemolytic reagent meets one or more of the above-mentioned calcium content criteria, and that the polyphenol concentration in the hemolytic reagent also meets one or more of the above-mentioned polyphenol criteria.

[0307] The inventors have found that the concentrations of some saponins and SPS in the hemolytic reagent can affect the stability of the hemolytic reagent, for example, through phase separation, which can lead to problems such as an increase in the turbidity or precipitation of solid particles over time. In fact, for example, when a high concentration of saponin in the hemolytic reagent, such as 3.75% to 7.50% w / w (for example, approximately 37.5 to 75 mg / mL in a 2 mL volume of hemolytic reagent), is present along with a high concentration of sodium polyanethole sulfate (SPS) of 2.50% to 4.00% w / w (or approximately 25 to 40 mg / mL in a 2 mL volume of hemolytic reagent), a small amount of precipitate (e.g., 0.1 mg to 1 mg in a 5 mL solution) may form over a period of one month at room temperature. Although not intended to be theoretically bound, this precipitate may be due to, for example, the interaction of SPS with the saponin itself or to impurities in the saponin material, such as polyphenols and polysaccharides, and is thought to form an insoluble complex. The inventors have discovered that the higher the temperature, the more the dissolving reagent is preserved and the faster the formation of this complex occurs. In a 2 mL collection container or device such as a saponin and SPS-containing hemolytic reagent, some insoluble material may form and potentially migrate and remain even after one or more separation steps (e.g., one or more rounds of centrifugation and precipitation).

[0308] It has been found that when such high concentrations of saponins and SPS are present, adding methyl-β-cyclodextrin (MBCD) to the hemolytic reagent prevents phase separation and precipitation during storage. For example, in a hemolytic reagent containing up to 7.50% w / w (approximately 75 mg / mL in a 2 mL volume) of saponins and up to 4.0% w / w (approximately 40 mg / mL in a 2 mL volume) of SPS, adding MBCD at a final concentration of 6.25 mM prevented any visible precipitation for at least one month at 45°C, whereas without MBCD, a considerable amount of precipitation was visible after two days at 45°C. It is thought that MBCD stabilizes other components in the solubilating reagent by encapsulating them and preventing their interactions and the formation of any insoluble complexes.

[0309] While not intended to be constrained by theory, methyl-β-cyclodextrin (MBCD) is thought to be more resistant to solubilization by other detergents and has the ability to permeate compartments of the cell membrane, thus potentially aiding in hemolysis. From a simple point of view, detergents function by intercalating into lipid membranes, rearranging membrane components to accommodate the increasing concentration of detergent, and then ultimately extracting lipids and proteins to form smaller micelle structures. The various fluid states of the lipid membrane affect the extent to which the detergent disrupts and stabilizes cellular components. Loosely packed membrane domains, made from twisted unsaturated glycerophospholipids and fatty acids, diffuse very easily and permeate readily with neutral detergents, causing liquid disorder (L d This leads to a state where lipids are ordered (L). However, some lipids associate with each other and perform lipid ordering (L oThese tend to form more tightly packed domains, resulting in a state where the lipid raft domains are more readily formed. These domains are composed of more hydrophobic lipids, namely cholesterol and sphingolipids, as well as saturated fatty acids and glycerophospholipids, and can resist permeation and extraction by detergents. These detergent-resistant membranes (DRMs), which are thought to give rise to lipid raft domains, do not dissolve completely in most detergents, leaving behind insoluble fragments of sphingolipids and cholesterol, as well as membrane proteins embedded in these fragments. The effect of these DRMs in human red blood cells is incomplete hemolysis due to a heterogeneous mixture of hemolytic reagents and solubilized proteins and lipids with insoluble cell debris.

[0310] Destruction of the DRM can be achieved by extracting cholesterol from within the lipid bilayer. Quillaja saponins are known to solubilize cholesterol by being incorporated into saponin micelles. During hemolysis, the saponins undergo micelle rearrangement with cholesterol within the cell membrane, leading to the formation of open pores that cause overall cell destruction. Beta-cyclodextrin (β-CD) also has a high affinity for cholesterol and is used to extract cholesterol from red blood cells and other membranes. The mechanism involves the association of CD dimers to the membrane surface, followed by the extraction of cholesterol dimers into hydrophobic cavities. The cholesterol-CD complex then desorbs from the membrane surface into the surrounding solution, where it can transfer cholesterol to another domain of the cell membrane or to potentially waiting detergent micelles, thereby carrying cholesterol away from the cell. The loss of a large portion of cholesterol from the membrane destabilizes the DRM, making it more susceptible to solubilization by other neutral detergents such as saponins. This results in more complete hemolysis without interference from solid debris.

[0311] While the inventors have shown that including MBCD is beneficial to reduce or remove precipitates during storage of saponin and SPS-containing hemolytic reagents, other β-cyclodextrins may be used in addition to or instead in some implementations. By conducting experiments at various concentrations and observing the presence or absence of precipitates in the hemolytic reagents during storage, those skilled in the art can determine suitable concentrations of one or more β-cyclodextrins.

[0312] Therefore, in some exemplary implementations, blood lysis reagents containing saponins and SPS may also contain methyl-β-cyclodextrin (MBCD) at concentrations of 0.1 mM to 20 mM, 0.5 mM to 10.0 mM, or 1 mM to 8 mM.

[0313] In some exemplary implementations, blood lysis reagents containing saponins and SPS may also contain methyl-β-cyclodextrin (MBCD) at concentrations of 1–5 mM per 3% w / w saponin, 2–6 mM per 4% w / w saponin, 3–7 mM per 5% w / w saponin, 4–8 mM per 6% w / w saponin, or 5–9 mM per 7% w / w saponin.

[0314] The inventors observed that insufficient mixing of blood and hemolytic reagent immediately after blood collection may be the cause of filamentous structure formation, which is evident in the pink appearance of the final eluted (cell suspension at the end of the sample processing procedure). In one implementation, this problem is prevented by adding an anticoagulant such as ethylenediaminetetraacetic acid (EDTA) or its dipotassium salt (K2EDTA) to the hemolytic reagent. This additive combines with calcium ions in the blood, which are cofactors for many enzymatic reactions in the coagulation cascade. SPS, also present in solubilants, is a known anticoagulant, but the activity of SPS is more driven by direct interaction with polycationic enzymes involved in coagulation, rather than binding to calcium. In fact, K2EDTA is used in blood tubes for whole blood collection, particularly by inhibiting the coagulation process. These tubes are designed to yield a final EDTA concentration in the range of 1.4–2.2 mg / mL when combined with blood. Since EDTA is known to have antibacterial properties, its concentration must be kept sufficiently low to ensure the viability of microbial cells before sample processing.

[0315] To determine the appropriate level of K2EDTA to be added to hemolytic reagents, the following experiment was performed. Four different hemolytic reagents were prepared, each containing 0.20% Tergitol® L-62, 6.25 mM MBCD, 4.25% SPS, and 5.00% saponin in 150 nM sodium acetate buffer, but each containing different amounts of K2EDTA (0 mg, 4 mg, 8 mg, or 16 mg) per 2 mL of reagent. 2 mL of each reagent was then filled into individual tubes, which were used as blood collection devices. Approximately 6–7 mL of whole blood from a healthy human donor was then added to each tube. Immediately after blood collection, each tube was inverted twice (instead of the preferred 10 times) to mix the blood with the lysis reagent. Furthermore, to promote coagulation, each tube was stored at 4°C for 4 hours, after which 6.5 mL of each mixture was removed and combined with 2 mL of 0.15 M Tris-based activator in a centrifuge tube.

[0316] Next, the mixture was subjected to centrifugal washing according to the method of Example 18. A photograph of the tube before removing 1.9 mL of supernatant is shown in Figure 24B. A reddish pellet is present at the bottom of the tube, which is observed when using a hemolytic reagent lacking EDTA. In contrast, in the case of a hemolytic reagent containing K2EDTA, the visible signs of pellet (the signs of the filamentous structure described above) are not apparent. After removing the supernatant, the remaining 100 μL of eluate was examined under a microscope. 1 μL of the liquid was distributed onto an agar plate, allowed to diffuse naturally and air dry, and then imaged with a 2x objective lens.

[0317] A typical photograph is shown in Figure 24C. As observed, undigested coagulation material was present in the case of hemolysis without K2EDTA, but the addition of K2EDTA in all quantities resulted in a more homogeneous distribution of particulate debris. Repeated testing with 38 blood donors revealed that the undesirable microcoagulation effect was donor-dependent. Visual inspection of the final eluate for signs of microcoagulation (Figure 24D) showed that microcoagulation was found in 32% of cases with hemolysis reagents lacking K2EDTA, while microcoagulation was prevented by supplying just 2 mg / mL of K2EDTA. Therefore, using K2EDTA in the collection tube reduces the risk of incomplete centrifugation during cell isolation from large volumes of blood.

[0318] In addition to functioning as an anticoagulant in the dissolving reagent, K2EDTA can also sequester naturally occurring calcium in saponins, which can lead to increased reagent instability, such as increased turbidity, before and after combination with alkaline activators. Under certain circumstances, calcium derived from saponins can also precipitate in blood coagulation, resulting in a red cell suspension. Therefore, controlling the amount of K2EDTA in the dissolving reagent can serve two purposes: providing reagent stability and acting as an additional anticoagulant in conjunction with SPS.

[0319] It will be understood that adding a wide range of chelating agents to the sample collection tube can promote the chelation (sequesteration) of calcium and other ions that may be present in the sample. A non-exclusive list of exemplary chelating agents includes CDTA (1,2-cyclohexylenedinitrilotetraacetic acid), DTPA (diethylenetriaminepentaacetic acid), EGTA (ethylenebis(oxyethylenenitrilo)tetraacetic acid), HEDTA (hydroxyethylethylenediaminetriacetic acid), NTA (nitrilotriacetic acid), GLDA (tetrasodium glutamate diacetate), EDDS (ethylenediamine-N,N'-disuccinic acid), MGDA (N-(1-carboxyethyl)iminodiacetic acid), IDA (iminodiacetic acid), BAPTA (1, Examples include 2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, EDDHA (ethylenediamine-N,N'-bis(2-hydroxyphenylacetic acid)), disodium citrate, and potassium oxalate. Most of these exemplary materials belong to a class of chelating agents called aminopolycarboxylic acids. These agents employ a coordination chemistry similar to EDTA, binding to ions, but many are not considered anticoagulants. They can be used in various initial salt forms that provide the best solubility.

[0320] Furthermore, a non-limiting list of exemplary substrates for K2EDTA that function as anticoagulants includes K3EDTA, disodium citrate, potassium oxalate, SPS, and heparin (sodium heparin, lithium heparin, or ammonium heparin).

[0321] Referring again to the flowcharts shown in Figures 18 and 19, it will be understood that directly isolating microbial cells from whole blood samples enables a variety of downstream applications, including, for example, nucleic acid amplification, sequencing, MALDI identification, antimicrobial susceptibility testing, and other applications. In some exemplary embodiments, microbial cells isolated from whole blood (or other sample types such as those described above) according to the methods described in Figures 18 and 19 or variations thereof can be used to grow and detect microcolonies according to any of the exemplary embodiments described above, and optionally process the microbial cells recovered from the microcolonies.

[0322] In some exemplary embodiments, at least a portion of the isolated microbial cells can be used for detection by molecular amplification assays in a direct format without the need for or the implementation of an intervening growth step. For example, isolated microbial cells can be optionally lysed and detected without performing nucleic acid extraction by a suitable nucleic acid amplification method, such as one of those described above. The amplification product can be detected directly and / or used to generate a next-generation sequencing library, for example, as described above. In fact, microbial cells isolated from whole blood samples (or other sample types, such as those described above) can be processed using one or more of the nucleic acid amplification and / or sequencing methods and workflows described above, associated with the use of microbial cells derived from recovered microcolonies, according to the methods described in Figures 18 and 19 or variations thereof. [Examples]

[0323] To enable those skilled in the art to understand and implement embodiments of the present disclosure, the following examples are provided. These should not be understood as limiting the scope of the present disclosure, but merely as examples and representative of these compounds.

[0324] Example 1: Summary of experimental research and results Bacterial suspensions were prepared in 1 mM PB buffer at approximately known concentrations and tested with rRT-LAMP according to the method of Example 9. Figure 7 shows that the assay sensitivities for clean samples against Escherichia coli ATCC 35218 and Staphylococcus aureus ATCC 25923 were 10 and 100 CFU, respectively. In cell suspensions obtained from whole blood samples, the detection limit is further increased due to assay inhibition by settling blood debris.

[0325] After measuring the sensitivity of the rRT-LAMP assay, 4-hour-old microcolonies were collected by biopsy and the washing method of Example 8, and rRT-LAMP was performed according to the method of Example 9. The results shown in Figure 8 demonstrate the equivalence of the two collection methods. Furthermore, the positive reporting time was less than 10 minutes. This type of TTP is well below the TTP resulting from environmental contamination.

[0326] The assay was also performed using the microcolony recovery exchange method according to Example 8D. The resulting TTP (average of 3 replications) for 10 different bacterial species (4 Gram-negative: Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumanni, Klebsiella pneumoniae; 6 Gram-positive: Staphylococcus aureus, Streptococcus pyogenes, Enterococcus faecalis, Staphylococcus epidermidis, Streptococcus pyogenes, Staphylococcus heamolyticus) is shown in Figure 9A. Here again, the measured TTP was in the range of 10 minutes or less, demonstrating the robustness of the test against environmental contamination.

[0327] The suitability of microbial cell recovery from microcolonies for downstream molecular assays was also demonstrated by performing PCR assays. In this case, the rRNA gene of genomic DNA was selected as the target, and specific PCR primers for Staphylococcus aureus ATCC 25913 were designed. Microcolonies at 4 hours and 6 hours of age were recovered using the swab method of Example 8C, and cell suspensions were prepared in 1 mM PB buffer. As a negative control, an area of ​​a plate without microcolonies was swabded using a method mimicking that of Example 8C. The cell suspensions were lysed according to the method of Example 10 and tested by PCR assay. The results are shown in Figure 10A.

[0328] In another study, microcolonies were collected, grown for 4 hours using the exchange method of Example 8C, and placed in 200 μL of PB buffer. The cell suspension was divided into two 100 μL aliquots. A PCR assay was performed on both aliquots according to the method of Example 10, with one aliquot undergoing thermal lysis and the other not lysed before adding the sample with the master mix. The results shown in Figure 10B show that the two assays performed similarly, as quantified by CT values. Here again, this unexpected observation is beneficial, considering that the assay method is simplified by avoiding any form of cell lysis, and the likelihood of interfering events such as target loss and / or introduction of inhibitors and contaminants occurring during the cell lysis step is reduced. In addition, glass bead disruption, which is often used for lysing Gram-positive cells, shears the gDNA and breaks it into smaller pieces. In contrast, the integrity of the gDNA is maintained by avoiding cell lysis or using a milder lysis approach, such as thermal lysis or electrolysis. This is particularly useful for sequencing applications.

[0329] Example 1A: Rapid Gram staining of microbial cells recovered from microcolonies Affinity surfaces for recovering microbial cells from microcolonies were prepared by cutting from the slides mentioned, and their effectiveness was verified by Gram staining according to the method of Example 7B for all bacterial targets listed in Figure 4. Figure 5 shows the results for one sample in the case of E. coli microcolonies grown for 4 hours after diffusion of the cell suspension. Qualitatively, in terms of transfer efficiency, Fisherbrand® Superfrost® Plus Microscope Slides (Control SF), Rat tail Collagen I (collagen), mouse laminin (laminin), and gelatin were equally good. Among these, laminin tended to be more susceptible to staining artifacts such as false staining. Cells transferred by the PLL-coated surface did not disperse for Gram staining.

[0330] Another embodiment of the affinity surface was prepared according to the method of Example 7A. The results for Staphylococcus aureus microcolonies are shown in Figure 6.

[0331] Example 2: Preparation of microbial cell culture medium Excluding Staphylococcus aureus and Streptococcus pneumoniae cell cultures, Gram-positive bacteria were prepared as follows.

[0332] 1.30 μL of the corresponding bacterial species and staining glycerol stock solution were seeded onto 3 mL of triptych soy broth (TSB), and incubated overnight at 37°C with shaking at 150 rpm.

[0333] 2. A 10-fold dilution of the culture solution in TSB was incubated at 37°C for 1 hour (Enterococcus faecalis, enterococci, and Streptococcus agalactiae) or 2 hours (Staphylococcus epidermidis).

[0334] Gram-negative bacteria, excluding Pseudomonas aeruginosa cell culture, were prepared as follows.

[0335] 1.30 μL of the corresponding bacterial species and staining glycerol stock solution were seeded onto 3 mL of TSB and incubated overnight at 37°C with shaking at 150 rpm.

[0336] 2. A 10-fold dilution of the culture solution in TSB was incubated at 37°C for 1 hour (Acinetobacter baumanni, Enterobacter cloacae complex, Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis) or 2 hours (Serratia marcescens).

[0337] Staphylococcus aureus cell culture medium was prepared as follows.

[0338] 30 μL of the corresponding staining glycerol stock solution was seeded onto 3 mL of TSB and incubated at 37°C for 3 hours with shaking at 150 rpm.

[0339] The pneumococcal cell culture medium was prepared as follows.

[0340] 30 μL of the corresponding seed or stained glycerol stock solution was seeded into 3 mL of TSB and incubated at 37°C for 3 hours with shaking at 80 rpm in the presence of a CO2 generating pouch.

[0341] Pseudomonas aeruginosa: 1.6 μL of Pseudomonas aeruginosa strain glycerol stock solution was streaked on a 5% sheep blood plate with tryptic soy agar (TSA) and incubated overnight at 37°C (P1).

[0342] 2. Bacterial colonies were subcultured on agar plates at least twice (P2).

[0343] 3. Colonies were seeded from the plate into 3 mL of TSB and incubated at 37°C for 3 hours with shaking at 150 rpm.

[0344] Based on OD measurements, serial dilutions of each bacterium were prepared in TSB at a nominal concentration of 1000 CFU / mL.

[0345] Example 3: Preparation of agar-based solid-phase growth medium plate Agar-based solid-phase growth plates were prepared with a final agar concentration within the range of 1.75% w / v.

[0346] Base / anhydrous culture medium composition (BD to BBL2): Pancreatic digest of casein; 14.5 g / L, papain digest of soy diet; 5 g / L, sodium chloride; 5 g / L, agar; 14 g / L, growth factor; 1.5 g / 4 g of BBL2; 0.35 g of agar; 95 mL of distilled water; 5 mL of defibrinated sterile sheep blood.

[0347] The preparation steps were as follows:

[0348] 1. Heat the TSAB powder in molecular biology grade water on a hot plate at 100°C for 10 minutes in a water bath.

[0349] 2. Add TSAB to the water.

[0350] 3. Heat to boiling point and stir thoroughly.

[0351] 4. Autoclave the solution (at 120°C for 15 minutes).

[0352] Cool to 5.55°C, add blood, and mix thoroughly.

[0353] 6. Preheat the pipette in warm water.

[0354] 7. Distribute onto plates and allow to solidify for 5 minutes.

[0355] Example 4A: Microcolony growth in solid growth medium Distribute the cell suspension from Example 3 onto the plate from Example 4 (a plate with a diameter of d=55 mm) and spread for approximately 10 cm.2 The sample was diffused over the area using an L-shaped spreader (Global Scientific). The plate was incubated in an in-house incubator (T=36°C, saturated humidity) and scanned every 30 minutes with a metallurgical microscope using a 2x objective lens.

[0356] Example 4B: Diffusion Spiral smear Diffusion in spiral smearing uses an instrument designed to distribute a liquid sample in a spiral pattern into a Petri dish. The spiral smearing device rotates the dish (or moves the dispensing tip) while simultaneously distributing the liquid and moving the dish or dispensing tip linearly. Diffusion can be carried out as follows:

[0357] 1. Fill the dispensing chip with the concentrated bacterial suspension to be dispersed.

[0358] 2. Place the distribution chip in the center of the agar plate.

[0359] 3. While rotating the agar plate, simultaneously distribute the liquid and move the distribution tip in a straight line from the center of the agar plate towards the periphery.

[0360] L-shaped smearer Dispersion can occur as follows:

[0361] 1. Using a micropipette, place the concentrated bacterial suspension in the center of the agar plate without touching the agar.

[0362] 2. Using a sterilized smearing device (L-shaped), gently rub the surface of the agar while rotating the plate with one hand.

[0363] 3. The liquid continues to diffuse until it is absorbed by the agar.

[0364] 4. After diffusion is complete, remove any residue from the smear container with alcohol or bleach, and discard the smear container into a waste beaker.

[0365] Example 4C: Image registration in the spatial frequency domain 1. Convert the image to binary using the Otsu method.

[0366] 2. Perform separate Fourier transforms (DFRs) on the two images.

[0367] 3.Calculate the complex conjugate of the DFT of the second image.

[0368] 4. Multiply the complex conjugates of the DFT of the 4.1 image and the DFT of the 2nd image. This operation is also known as the cross-power spectrum.

[0369] 5. Next, the inverse discrete Fourier transform (IDFT) is calculated from the cross-power spectrum, which yields the cross-correlation in the spatial domain.

[0370] 6. The position of the peak in the cross-correlation corresponds to the substitution of the two images.

[0371] Example 5: Detection and localization of microcolonies The image registration step was used for microcolony detection. By aligning consecutive images, the object detection algorithm compares and matches the features of the objects (blood debris), resulting in more accurate and reliable detection results. To align imaging data obtained at various points in time, 2D-2D registration (with limitations of affine transformation) was performed on each image. Image registration was based on the detection and extraction of local characteristic features such as corners, boundaries, and / or stains. A SURF (Speeded-Up Robust Features) detector was used to extract features from 2D grayscale input images. Two images of the same scene taken at different times were aligned during image registration. The goal was to discover a transformation that maps pixels in one image to corresponding positions in the other image, based on prior knowledge. The transformation obtained by incorporating prior knowledge into image registration was more accurate and robust. Intensity traits present in one or both were classified as background, while intensity traits appearing in subsequent images were classified as foreground. Objects with more than 250 pixels present in the foreground were classified as microcolonies. Recorded candidate locations of detected microcolonies were used as the site locations.

[0372] Example 6: Preparation of affinity surfaces for microcolony recovery This embodiment presents a method for coating a glass surface that can be used as an affinity surface for microcolony recovery.

[0373] Materials used: 1 L of deionized H2O, 5 g of gelatin (G6144-100G gelatin derived from pig skin, Sigma-Aldrich), and 0.5 g of potassium chromium sulfate dodecahydrate CrK(SO4)2·12H2O (243361-5G potassium chromium(III) sulfate dodecahydrate, Sigma-Aldrich).

[0374] Preparation steps: 1.1 L of deionized H2O was heated to 45°C.

[0375] 2.5g of gelatin was added and mixed with a magnetic stirrer until the gelatin crystals disappeared.

[0376] 3.05 g of potassium chromium sulfate dodecahydrate was added, and stirring was continued until the solution turned pale blue.

[0377] 4. (Optional step): Add a few crystals of thymol as a preservative.

[0378] 5. Cool the solution to room temperature.

[0379] 6. The solution was filtered to remove any large particles (if any).

[0380] 7. Place the microscope slide (affinity surface requiring coating) into a suitable side holder (e.g., Thomas Scientific part number 8542K20).

[0381] 8. (Optional step): Rinse and moisten the slide with warm water.

[0382] 9. (Optional step): Place the slide in warm, foamy water for 10 minutes, stirring if necessary.

[0383] 10. (Optional step): Rinse the slide with warm water until all traces of bubbles are gone (about 15 minutes).

[0384] 11. (Optional step): Rinse the slide with distilled water while swirling it around (for about 5 minutes).

[0385] 12. (Optional step): Immerse the slides in 70% EtOH. Remove any excess ethanol and let them dry overnight in a dust-free area. Place the slides in a rack with the absorbent paper facing downwards and cover with a large container or tin foil.

[0386] 13. Immerse a rack of clean slides in a warm gelatin solution (40-50°C) for 3-5 minutes (approximately 5 seconds each time).

[0387] 14. Remove the rack containing the slides and soak the excess gelatin.

[0388] 15. Allow the slides to air dry for 24 hours in a dust-free environment.

[0389] Example 7: Gram staining of cells recovered from microcolonies material: Gram staining kit: Gram crystal violet, Gram iodine or stabilized Gram, iodine, Gram dechromizer, and Gram safranin. (Cat. No 212539, BBL, BD).

[0390] procedure: 1. Gently press the affinity surface onto the region containing the detected microcolony.

[0391] 2. Move the surface and allow it to air dry.

[0392] 3. By flowing absolute methanol onto the surface (for 1-2 minutes), the methanol is fixed to the affinity surface.

[0393] 4. Allow the affinity surface containing the microcolonies to air dry.

[0394] 5. Apply the primary dye (Gram Crystal Violet) to the affinity surface and wait for 1 minute.

[0395] 6. Remove the primary dye by immersing the affinity surface in cold water and gently stirring.

[0396] 7. Immerse the slide in the mordant (stabilized Gram iodine) and wait for 1 minute.

[0397] 8. Remove the mordant by immersing the affinity surface in cold water and gently stirring.

[0398] 9. Immerse the affinity surface in the Gram Dashboard and wait 30 seconds.

[0399] 10. Immerse the affinity surface in cold water to clean it.

[0400] 11. Immerse the affinity surface in counterstain (Gram safranin) and wait 60 seconds.

[0401] 12. Immerse the affinity surface in cold water to clean it.

[0402] 13. Let it air dry.

[0403] The affinity surface is investigated under an oil immersion lens (100x magnification).

[0404] Example 8: Example of a microcolony recovery method: The following examples illustrate some non-limiting specific implementations of the recovery method described above.

[0405] Example 8A: Example of a biopsy method for microcolony recovery This method involves extracting / biopsing a small portion of the solid growth medium containing the detected / located microcolonies using a very small biopsy punch (or pipette tip as an alternative). An exemplary implementation of this method is described below.

[0406] Use a pipette with a 20-200 μL tip pre-filled with 1,100 μL of phosphate buffer (PB).

[0407] 2. Lower the chip as close as possible to the microcolony.

[0408] 3. Press the tip downwards to cut (not squeeze) the gel. / The cut portion of the gel will move inward towards the tip.

[0409] 4. Move the tip upwards and distribute the cut microcolonies into the tube along with the PB.

[0410] 5. After vortexing the tube for 10 seconds, separate the solid growth medium material (cylinder) from the PB (pipette contents) by simple centrifugation or coarse filtration.

[0411] The remaining PB is the final cell suspension.

[0412] Example 8B: Example of a washing method for microcolony recovery This involves washing the bacterial cells from the gel surface with a buffer consistent with the downstream assay. An exemplary method is as follows:

[0413] Aspirate 1.10 μL of buffer solution.

[0414] 2. Bring the detected / located microcolonies to the chip and hold them near the surface.

[0415] 3. Distribute the buffer solution.

[0416] 4. Immediately bring the surface into gentle contact with the chip.

[0417] 5. Scrap the surface to remove bacterial cells.

[0418] 6. Aspirate the buffer solution.

[0419] 7. Transfer the contents to a sterile tube.

[0420] Example 8C: Example of a swab method for microcolony recovery The exemplary swab method used was the VITEK® PICKME® Pen (bioMerieux).

[0421] 1. Use the PickMe pen to pinch and extend the pen tip.

[0422] 2. Bring the pen tip over the detected / located microcolony.

[0423] 3. Slowly bring the pen tip into contact with the area.

[0424] 4. Move the pen tip over the surface several times.

[0425] 5. Move the pen tip into the tube containing the buffer suitable for the downstream assay.

[0426] 6. Stir the pen tip to release the cells.

[0427] 7. Extend the pen tip and discard it.

[0428] Example 8D: An example of aspiration using a conductive pipette tip. One implementation of microcolony retrieval by aspiration involves using a conductive pipette tip, which ensures precise positioning of the tip relative to the gel surface. The steps are as follows:

[0429] 1. Detect and place microcolonies.

[0430] 2. Pick out the conductive pipette tip.

[0431] Aspirate 3.5 μL of 1 mM PB buffer.

[0432] 4. Place the chip on top of the microcolony.

[0433] 5. Feedback from electrical positioning detection ensures the actual placement of the chip on the gel surface.

[0434] 6. Lower the tip by approximately 0.3 mm and press to ensure good sealing with the gel surface.

[0435] 7. Dispense the recovered buffer into the gel.

[0436] 8. Aspirate the distributed recovery buffer to draw the microcolonies into the chip.

[0437] 9. Microscopic examination of the microcolony locations verifies the success of the retrieval.

[0438] Example 8E: Example of an electrostatic method for microcolony recovery An example of a retrieval method using an electric pin is as follows:

[0439] 1. Select the pins and apply an electrical insulation layer of 1 Mil Kapton® HN film.

[0440] 2. Connect the pin to the positively charged electrode.

[0441] 3. Bring the pin over the microcolony.

[0442] 4.V g Apply 18V (to the gel).

[0443] 5. Lower the pin to bring it into contact with the gel surface containing the microcolony, and keep the microcolony in contact with the pin for at least 10 seconds.

[0444] 6. Bring the pins onto the substrate or buffer solution where the microcolonies will be distributed.

[0445] 7.V s Apply -12V (to the substrate or buffer).

[0446] 8. Lower the pins to make contact with the circuit board or buffer.

[0447] Example 8F: Investigation of the reproducibility of microcolony recovery The following experiment was conducted to investigate the reliability of the suction method for recovery.

[0448] 1. Receive target microbial cells (106 CFU / mL in TSB) from the cell culture medium and prepare serial dilutions to 104 CFU / mL in TSB for spiking.

[0449] Spike 2.5 μL (nominal 50 CFU) into 100 μL of culture medium.

[0450] 3. Diffuse the cell suspension onto an agar plate.

[0451] Incubate in an incubator at 4.35°C.

[0452] 5.1 Imaging is performed every hour to detect microcolonies and measure their locations.

[0453] After 6.5 hours of incubation:

[0454] (a) Randomly select one microcolony and collect it in 5 μL of physiological saline.

[0455] (b) For the plate on which to count the number of biomass, distribute the collected material in a microtube into 100 μL of physiological saline (a 1 / 20 dilution of one colony pinch) and resuspend it.

[0456] (c) The location of the microcolony is imaged to confirm that the operation was successful.

[0457] (d) Repeat the above steps for two or more microcolonies.

[0458] 7. In step 6(b), dilute the cell suspension to a 1 / 100 or 1 / 1000 dilution of a single colony pinch.

[0459] Plate each of the 8,100 μL of Step 8 in three different ways.

[0460] 9. Incubate the plate overnight.

[0461] 10. Count the number of colonies and calculate the total biomass per colony pinch.

[0462] 11. Investigate the biomass of microcolonies and its deviations using three different methods.

[0463] Example 9: Real-time RT-LAMP assay Cells were lysed by heating the cell suspension to 95°C for 10 minutes using a GeneAmp® PCR System 9700 (thermal lysis method). 5 μL of the lysate was added to the reaction well of a LightCycler® 96 Instrument cartridge, and the RT-LAMP enzyme (New England Biolabs, WarmStart® RT-LAMP Lyo-Ready kit) was dried at the bottom. After rehydration, the liquid was transferred to another well on the plate, and the mixture containing the STYOTM9 dye and the set of RT-LAMP target-specific primers were dried at the bottom. The plate was sealed and placed inside the LightCycler® 96 Instrument, heated to 64°C, and the temperature was fixed and maintained for 20 minutes. The time to positive (TTP) was defined as the time it took for the dynamic fluorescence signal to exceed the background.

[0464] Example 10: Real-time PCR assay Cells were lysed by heating the cell suspension to 95°C for 10 minutes using a GeneAmp® PCR System 9700 (thermal lysis) or by the electrolysis method described in Example 11. A 1 μL portion of the lysate was combined with 4 μL of a master mix containing STYOTM9 dye, a set of target-specific PCR primers (targeting the rRNA gene), and polymerase enzyme (Kappa HotStart, Roche). The mixture was pipetteed into a PCR plate, which was then placed in a LightCycler® 96 Instrument. 38 PCR cycles consisted of 30 seconds of growth (67°C) followed by 5 seconds of thawing at 95°C. CT values ​​were measured by the instrument based on real-time fluorescence signals.

[0465] Example 11: Electrolysis of cell suspension The electrolysis method used in this embodiment was the e-Lysis® method developed by Qvella®. The technology is described in international patent application PCT / CA2012 / 000698. Briefly, the lysis chamber has a volume of 20 μL and a thickness of 200 μm, including the upper and lower electrodes. The electrodes are formed from microstructured aluminum with a conformal aluminum oxide dielectric layer, as described above. After filling the chamber with the cell suspension, a series of square wave AC pulses with a duration of 50 μs and an amplitude of 200 V were applied for a duration of 50 ms.

[0466] Example 12: Dependence on surface artifact density in blood lysis reagent compositions To demonstrate the dependence of surface artifact density on the composition of hemolytic reagents, 4 mL whole blood samples were treated according to the method of Example 13, both undergoing two washing cycles with hemolytic reagents having the following compositions: (ii) 35 mg / mL saponin, 20 mg / mL SPS (BLR1), and (ii) 35 mg / mL saponin, 20 mg / mL SPS, 0.3% w / v Triton® X-100, and 50 mM carbonate-bicarbonate buffer (pH 10) (BLR2). After exposing the samples to the corresponding hemolytic reagents and centrifugation, 1 μL of each final microbial cell suspension was pipetteed onto a spot-on agar plate. The microbial cell suspension samples were diffused into circular areas with a diameter of approximately 5 mm and air-dried for approximately 3 minutes. These areas, labeled herein as mini-culture regions (MCRs), were imaged with a microscope equipped with a 5x objective lens and are shown in Figures 11A and 11B. In these figures, the MCR, the unused agar plate surface, and the boundary between the two regions are indicated by labels 310, 312, and 311, respectively. It is observed that the background (density of surface artifacts) is significantly reduced by including Triton X-100 and carbonate-bicarbonate buffer.

[0467] The inventors found that this background level could not be further significantly reduced by increasing the number of wash cycles. This was demonstrated, for example, by treating a 4 mL whole blood sample with two or four subsequent centrifugal washing cycles using BLR2 having the above formulation. 1 μL of the resulting microbial cell suspension was distributed onto an agar plate, diffused, and air-dried. Photographs of the resulting MCR were recorded with a 10x microscope objective lens, and the particle size distribution of the debris was analyzed at the end of the two and four wash cycles. The particles were positioned and fitted into ellipses using an intensity-based autothresholding method. More precisely, image segmentation was performed to assign each group of pixels in the image to a connection group such that pixels at the same level share specific intensity characteristics. The histogram distribution (major axis of the fitted ellipse) of the measured major particle sizes is shown in Figure 11C. As observed, despite the fact that the sample is diluted 400-fold between the two washes and the four washes, the distribution plots are qualitatively similar.

[0468] Example 13: Sample processing of 4 mL of spiked whole blood sample Sample preparation was performed on spiked whole blood samples as follows:

[0469] 4 mL of hemolytic reagent was added to 4 mL of spiked whole blood sample in a 1.15 mL centrifuge tube.

[0470] 2. The centrifuge tubes were mixed by vortexing at the maximum speed of the vortex for 1 minute.

[0471] 3. The centrifuge tube was centrifuged at 4000 rpm for 8 minutes.

[0472] 4.7.9 mL of the supernatant was removed.

[0473] 5.2.9 mL of washing buffer was added to the residue, the solution was mixed by gentle vortexing, and the mixture was centrifuged at 4000 rpm for 3 minutes. The first washing cycle was performed by drawing the solution and discarding 2.9 mL of supernatant so that 1000 μL of residual liquid was retained.

[0474] 6.2.9 mL of washing buffer was added to the residue, the solution was mixed by gentle vortexing, and the mixture was centrifuged at 4000 rpm for 3 minutes. The second washing cycle was performed by drawing the solution and discarding 2.9 mL of supernatant so that 1000 μL of residual liquid was retained.

[0475] 7.1.9 mL of washing buffer was added to the residue, the solution was mixed by gentle vortexing, and the mixture was centrifuged at 4000 rpm for 3 minutes. The third washing cycle was performed by drawing the solution and discarding 1.9 mL of supernatant so that 1000 μL of residual liquid was retained.

[0476] 8.1.9 mL of washing buffer was added to the residue, the solution was mixed by gentle vortexing, and the mixture was centrifuged at 4000 rpm for 3 minutes. The fourth washing cycle was performed by drawing the solution and discarding 1.9 mL of supernatant so that 1000 μL of residual liquid (cell suspension) was retained.

[0477] Example 13: Growth and detection of microcolonies To illustrate an example of the dynamic nature of microcolony formation based on microbial cells obtained directly from whole blood samples, 4 mL of whole blood samples were spiked and treated with 3000 CFU of Proteus mirabilis (PM) cells according to the method described in Example 12 below. 1 μL of the resulting cell suspension was distributed to each of four agar plates and allowed to naturally diffuse into circular areas with a diameter of approximately 5 mm, hereafter referred to as "mini-culture" areas (MCRs). A partial image of the resulting MCRs is shown in Figure 12. Three and four hours after the start of incubation, several microcolonies, indicated by arrows, can be observed by visual inspection of the images. However, to detect microcolonies during even shorter incubation times, e.g., within 2 hours, it may be necessary to analyze the differentiation of microcolonies from the background, indicated by arrows, in the images.

[0478] An exemplary method for microcolony monitoring is described below. As observed in Figure 13, 2D-2D registration (using translation and rotation) with strict transformation constraints was performed to align imaging data acquired at various time points (0, 2, 3, and 4 hours after seeding, as shown in the figure). Corresponding intensity feature points between the image at t0=0 and each subsequent image (t2=2 hours, t3=3 hours, t4=4 hours) were automatically identified using the keypoint detector SURF and used for alignment of imaging days relative to t0. Intensity traits present at t0 were classified as background, while intensity traits appearing in subsequent images (cells / bugs) were classified as foreground. Parts of a given individual microcolony were marked in sequential images.

[0479] Measuring the background allows for improved detection of microcolonies. For example, in the four images acquired in Figure 7, despite unusually large translation and rotation offsets, the colony identification at t3=3 hours after incubation is clear. This method facilitates the development of a fully automated microcolony identification (reduced time to positivity: TTP) and tracking system for screening image sequences of unstained live microorganisms. The background thresholding method described above (e.g., radius R>R) 閾値 =R back.av The robustness of the method, compared to estimation based on explicit identification of microcolony spots when +n*sd, is shown below in conjunction with the results presented in Figures 14A-14H.

[0480] The growth rate in solid growth medium (gel) was measured according to the steps of Example 14 below. The number of colonies forming units in the MCR was counted, and its logarithm (Log(CFU)) was plotted against incubation time for Proteus mirabilis (PM), as shown in Figure 15A. Using the slope of this curve, i.e., 0.97, the growth rate was calculated using the relationship: growth rate = slope / log(2) = 3.23 cycles / hour. As another example, the growth rate of Staphylococcus epidermidis (SE) was measured. The measured v(Log(CFU)) against incubation is shown in the time plot in Figure 15B. The calculated growth rate was 2.4 cycles / hour. This growth rate is approximately 1.5 times higher than the measured growth rate of Staphylococcus epidermidis incubated on a CMOS chip [Jung, Jae Hee, and Jung Eun Lee. "Real-time bacterial microcolony counting using on-chip microscopy." Scientific reports 6 (2016):21473]. Depending on the preparation method of the stock solution, seeded microbial cells may go through an induction phase before growing into microcolonies. For example, as observed from Figure 15C, Pseudomonas aeruginosa (PA) cells show a lag time of approximately 2 hours. A linear trend on a semi-logarithmic scale is expected to continue, although the number of cells in the colony is low enough that the majority of cells are capable of division. When the number of cells in the internal region of the microcolony, where there is no space for growth, exceeds the number of cells in the periphery, the overall growth rate of the microcolony is expected to decrease. As shown in Figure 15, in the case of E. coli, up to 10 6 Such deviations for microcolonies containing individual cells were not observed by the inventors. Therefore, it appears justifiable to use the growth rate and lag time to estimate the time required to reach the desired number of cells.

[0481] Figures 14A–14F show the measured time lag and growth rate of seeded cells, measured by microcolony detection, for populations of microbial cell species that constitute the majority of pathogenic microorganisms typically encountered in bloodstream infections. The table also includes measured cell recovery fractions, i.e., fractions of cells that were successfully separated from spiked blood samples and resuspended in cell suspension while maintaining viability, measured according to the method of Example 15. In addition, the table also shows the estimated time to positive (TTP) for exemplary growth methods involving colony growth on solid growth medium ("solid phase"), where microcolonies were measured for a time identifiable against the background.

[0482] TTP is affected by the sensitivity of the detection method and its associated analysis and background. In the simplest case, introducing the presence of microcolonies grown from microbial cells isolated from a whole blood sample using the simple size selection method described above, TTP can be estimated as follows: Parameters for two washes in Figure 11C: R 閾値 Using =2+3*1.5=6.5μm, the threshold size R 閾値 =R back.av +n*std was calculated. Dense packing and 1μm 2 Estimating the worst-case scenario of the average nearest size, if the radius is R 閾値 The number of cells within the circle is approximately 120 CFU. Therefore, TTP = T lag The result is +7 / growth rate.

[0483] In the case of fungal species, due to their larger size compared to bacteria, a single division resulting in binary fission may be sufficient to detect microcolonies and yield a positive measurement. This is shown in Figure 16, which shows a time-lapse image of a blood agar plate section into which a 1 μL microbial cell suspension containing microbial cells isolated from a whole blood sample has been distributed. As can be calculated from the growth rate data in Figures 9G-9H, after approximately 4 hours of incubation, the cell count has increased by about 3 times. The growth of fungal cells is easily recognizable by comparing the two images. Therefore, in large distributions of fungal cells, the time to positive is TTP=Tlag The conclusion is that +1 / proliferation rate. In typical blood samples, single-digit cell counts and Poisson statistics cannot be ignored. Therefore, the formula needs to be replaced with TTP = Tlag + n / proliferation rate, where n is greater than 1. In one exemplary implementation, the value n=2 was used.

[0484] The characteristic growth rate is comparable to that in the planktonic state. To demonstrate this agreement, the growth rate in liquid culture medium was estimated from experiments conducted as described below. Whole blood samples (10 mL) spiked with various strains of microbial cells at a concentration of 5 CFU / mL were seeded into corresponding BacT / ALERT® FA Plus culture bottles and incubated with BacT / ALERT® VIRTUO. After the incubator showed a positive result, 1 mL aliquots were taken from each bottle, serially diluted, and plated to measure the number of CFUs. Ignoring lag time and assuming a constant growth rate, the growth rate was estimated based on the initial spike concentration ratio, the final bacterial concentration at the time of positive plate counting, and the time to positive (TTP). As observed in Figures 14A-14H, the growth rate in solid growth medium and the growth rate in liquid growth medium are similar. However, as can be clearly understood from the TTP value, the solid phase is advantageous as a result of the localization nature of microcolonies, and the solid phase becomes easier to detect at a much earlier time. For example, most bacterial species are easily detectable 3 hours after plating according to the exemplary method of these microcolonies, but the TTP of incubation in the culture bottle is usually more than 10 hours.

[0485] Figures 14A to 14G are 10 4 ~10 5 This also includes estimating the time required for bacterial cells to produce microcolonies containing individual cells. As discussed below, these quantities are relevant to subsequent microbial identification and / or antimicrobial susceptibility testing.

[0486] To characterize the implementation of exemplary methods for the rapid and direct formation and detection of microcolonies, two characteristics of common pathogens found in bloodstream infections, namely (i) lag time and (ii) proliferation rate, were measured. Recovery fractions of these pathogens from blood samples were also measured.

[0487] As shown in Figures 17B and 17C, a selected size threshold (e.g., diameter threshold) can be used to ensure that a sufficient number of microbial cells are reliably collected for diverse cell classes (e.g., species). For example, as shown in Figures 17B and 17C, when collecting microcolonies after reaching a diameter threshold of approximately 50 μm, at least 10 3 Individual microbial cells can be obtained across a wide range of microbial cell species.

[0488] Similarly, as shown in Figures 17B and 17C, when collecting detected microcolonies after reaching a diameter threshold of approximately 150 μm (for example, before reaching diameters of 180, 190, 200, and 250 μm), at least 10 5 Individual microbial cells can be obtained across a wide range of microbial cell species. Although the exemplary threshold embodiment refers to diameter, it will be understood that other size indicators such as radius or area may be used instead.

[0489] Figure 17A plots the dependence of microcolony diameter on cell content in exemplary cases of E. coli (obtained according to the method described in Example 14). The scatter plot is fitted with an exponential trend line by calculating the average microcolony diameters at cell content of 103 and 105 cells to be 60 μm and 170 μm, respectively. Following a similar approach, 10 3 and 10 5 The average diameter at which CFU cells are contained was calculated for 17 types of pathogenic Gram-positive and Gram-negative bacteria, and the results are shown in Figures 17B and 17C, respectively. According to this information, when bacterial microcolonies are harvested when their diameter reaches 65 μm, the number of microbial cells in the microcolony is 10, regardless of its density.3 ~10 5 It is likely to be in the range of CFU.

[0490] Example 14: Measurement of the growth rate and colony size of microbial cells on an agar plate The growth rate of microbial cells on an agar plate is measured by the following steps.

[0491] 10 5 Prepare a cell suspension with a nominal concentration of 10 CFU / mL.

[0492] On an agar gel plate, dispense 1 μL of the cell suspension into one of three distinguishable regions, spread the suspension into the mini-culture region (MCR), and air dry it. Thus, there are three MCRs identified as MCR1, MCR2, and MCR3 on the plate.

[0493] Image the MCR at t0 = 0 hours.

[0494] Plate the plate at 37 °C for 1 hour.

[0495] Image MCR1 at the 2-hour time point for bacteria and at the 4-hour time point for fungal species.

[0496] Analyze the images to calculate the area of the microcolonies, and calculate the corresponding diameter D using the equation D = 2 * sqrt(area / 3.1416). Then, average all the microcolonies to calculate the average diameter.

[0497] Remove the microbial content of the MCR by swabbing and resuspend it in 200 μL of TSB growth medium (cell resuspension).

[0498] Serial dilute the cell resuspension 10 times in TSB, and label the resulting samples as S10 0 、S10 -1 、S10 -2 、S10 -3 and S10 -4 as.

[0499] Plate the sample and incubate overnight.

[0500] Repeat steps 5 - 9 three, four times for MCR2 and MCR3 respectively and optionally for 6 hours (5 and 6 hours for fungal species) for bacterial species. Count the number of colonies overnight and tabulate.

[0501] Therefore, count the number of microbial cells in the corresponding MCR.

[0502] Measure the growth rate by calculating the slope of the plot of cell number and time on a logarithmic-linear plot.

[0503] Plot the average colony diameter against the colony cell content.

[0504] Measure the average diameter at which the number of cells in the microcolony reaches 10 3 ~10 5 to.

[0505] Example 15: Measurement of recovery rate and colony formation of microbial cells isolated from blood samples on agar plates The recovery rate for spiked whole blood samples was measured as follows.

[0506] In a cartridge containing 4 mL of BLR of Example 4, a spiked whole blood sample (4 mL) (prepared according to Example 2).

[0507] Mix the blood sample and BLR five times.

[0508] Centrifuge the cartridge at 3000 g for 8 minutes.

[0509] Transfer 7.9 mL of the supernatant to the waste chamber of the cartridge.

[0510] Perform the first wash cycle by adding 2.9 mL of wash buffer to the residue and mixing the solution.

[0511] Centrifuge 3000g for 3 minutes.

[0512] 2.9 mL of the supernatant was transferred to the waste chamber.

[0513] Repeat steps 5-7 for the second wash.

[0514] Remove 100 μL of residue (cell suspension).

[0515] Plate the cell suspension onto an agar plate and incubate overnight at 37°C.

[0516] The number of colonies is counted against the predicted number on the control plate, and the recovery is calculated.

[0517] Example 16: Experimental staining method Microscopic imaging of the mixture was performed according to Wright's staining method, as follows: 1. Prepare a smear (film) of the mixture on a microscope slide and air dry it. 2. Place the air-dried slides in the slide staining rack with the smear side facing upwards. 3. Cover the smear with an undiluted staining solution to fix it in place and partially stain it. 4. Wait 2 minutes. 5. Add approximately the same amount of water. Wait 6.5 minutes. 7. Rinse the stained smear. 8. Dry the slides at room temperature and examine them using a 100x oil immersion microscope.

[0518] Example 17: Effect of calcium concentration on the performance of saponin-based blood lysis reagents. To demonstrate the effect of high calcium content in saponin materials, two solutions were prepared in 8.5 mL of water using 96 mg of each material, each with the same 86% w / w triterpene saponin content, as well as nearly equal polyphenol content (0.7-0.8% w / w) and polysaccharide content (3.8-4.0% w / w). However, one solution used material with 0.6% w / w calcium, while the other solution used material with 0.3% w / w calcium. The solution with the higher calcium content appeared cloudy, while the other solution with the lower calcium content was clear and transparent.

[0519] Centrifuge was performed at 4000 revolutions per minute for 3 minutes. In the high-calcium solution, particles causing turbidity were collected as a dark pellet at the bottom of the centrifuge tube, while no visible pellets were observed in the low-calcium solution. Subsequent manual washing with 1 mM phosphate buffer and centrifugation yielded 100-120 μL of each solution. One was dark and opaque due to calcium turbidity, while the other was clean and clear. This study illustrates how turbidity caused by calcium in saponin material is transferred to the final cell suspension when used in lysis reagents for whole blood processing.

[0520] To test the tolerance of calcium in dissolving reagents derived from saponin components, sets of stock saponin solutions were prepared by mixing various batches of saponin solutions while increasing the calcium concentration, varying the amount of calcium. Each reagent contained 96 kg of saponin material consisting of at least 85% w / w triterpene saponins, 0.7–0.8% w / w polyphenols, and 3.8–4.0% w / w polysaccharides, with 0.33%, 0.46%, and 0.79% w / w calcium by dry weight. Using these saponin solutions, different dissolving solutions containing 0.20% Tergitol® L-62, 6.25 mM MBCD, 4.25% SPS, and 5.00% saponin (the given stock solution), respectively, were prepared in 150 mM acetate buffer. The resulting 2 mL of blood lysis reagent contained 0.32, 0.43, 0.57, and 0.68 mg of Ca, respectively. These were filled into individual tubes. The higher the calcium concentration, the greater the turbidity of the solution over time in the reagent.

[0521] These tubes were used as blood collection devices. After collecting 7 mL of whole blood from each tube, the tubes were inverted 10 times to mix the blood with the lysis reagent. Next, 6.5 mL of each mixture was removed from the device and combined with 2 mL of 0.15 M Tris base activator in a centrifuge tube. After standard centrifugation and washing with 1 mM phosphate buffer, the resulting 100–120 μL of recovered cell suspension was grown on agar plates and observed under a microscope after 30 minutes.

[0522] Figures 22A–22D show typical sections of each plate. Corresponding to each frame, we provide indices of the measured average debris particle size and surface coverage (percentage of plate area obstructed by debris). As observed, both the average debris particle size and surface obstruction percentage (%) increase with increasing saponin Ca content. Although not bound by theory, it is presumed that particles formed by the interaction of Ca and blood components settle during centrifugation washing, becoming the final cell suspension.

[0523] As observed in Figures 22A-22D, a lower amount of calcium in the dissolving reagent corresponds to a smaller number and size of precipitated debris. This translates to improved microcolony detection and recovery. In one experiment, collected blood samples were spiked in various tubes (with different Ca concentrations as described above) containing nominally 40 CFU of E. coli cells. Each collected blood sample, 6.5 mL, was processed according to the standard method described in the Examples and diffused onto an agar gel. The gel surface was monitored during incubation. The number of detected microcolonies is shown in Figure 23. As observed, reagents with lower Ca content had better recovery rates and earlier detection times (time to positive: TP). According to this experiment, the preferred upper limit for calcium content in 2 mL of dissolving reagent is less than 0.78 mg. When processing smaller volumes of blood (e.g., 3 mL), it is understood that the acceptable amount of Ca may be 100 mg in 2 mL of dissolving reagent.

[0524] Example 18: Beneficial role of methyl-β-cyclodextrin in acidic blood lysis reagents. To prevent precipitation in the dissolving reagent and to measure the concentration of MBCD, various amounts were added to a solution containing 4.25% w / w SPS, 5.00% w / w saponin material, 0.20% w / w Tergitol® L-62, and 0.15 M sodium acetate. The final concentrations of MBCD were 0 mM, 3 mM, 6.25 mM, 9 mM, and 15 mM. Each 5 ml solution was then stored at either room temperature or 45°C. At 45°C, after one week, a white, fuzzy solid was suspended in the solution without MBCD, but with 3 mM MBCD, the amount of this solid was significantly reduced. No solid was observed in the solution at MBCD concentrations of 6.25 mM or higher. In the sets of solutions stored at room temperature, similar results were observed for both the 0 mM and 3 mM MBCD samples, except that the first observation of the solid was made after one month. Therefore, the preferred amount of MBCD required to stabilize a mixture of 4.25% w / w SPS and 5.00% w / w saponin is at least 3 mM in the lysis reagent packed in the collection tube. On the other hand, it has been observed that final concentrations of MBCD exceeding 9 mM affect the viability of some bacterial cells, resulting in a decrease in cell recovery in the final microbial cell suspension.

[0525] Example 19: Exemplary method for centrifugation of microbial cells The basicized mixture was mixed for 1 minute using a vortex shaker. The mixture was then centrifuged at 4000 RPM for 8 minutes. The supernatant was removed by pipetting, leaving 100–120 μL of cell suspension at the bottom of the conical tube. The suspension was then pipetteed up and down to resuspend the cells in 1 mM phosphate buffer (2.2 mL), and the suspension was washed by mixing it with a vortex shaker for 1 minute, followed by centrifuging at 4000 RPM for 3 minutes. The supernatant was removed, leaving 100–120 μL of cell suspension. This washing sequence was repeated each time using fresh 1 mM phosphate buffer (2.2 mL) to obtain a final 100–120 μL of clean cell suspension.

[0526] Example 20: Direct molecular detection in microbial cells isolated from whole blood. This embodiment describes experiments conducted to demonstrate the ability of hemolytic reagents and processing methods used according to the methods disclosed herein to isolate microbial cells from whole blood and to use the isolated microbial cells for direct identification by molecular amplification. Figure 24A is a table showing the composition of the exemplary hemolytic reagent used in the experimental demonstration of cell recovery and molecular amplification.

[0527] The recovery rate of organisms from whole blood was demonstrated using whole blood collected in BD Vacutainer® SPS tubes. Since the SPS tubes consisted of approximately 8 mL of liquid containing 80% whole blood and 20% SPS solution, the total volume of whole blood in the sample was approximately 6.4 mL. A first mixture was obtained by combining 6 mL of SPS-whole blood sample with 2 mL of hemolytic reagent (BLR-1, shown in Figure 24A). This first mixture was then spiked with approximately 50 CFU of microbial cells from various microbial species. The first mixture was then left to allow a time delay of approximately 30 minutes at room temperature, after which alkaline buffer was added (according to the method in Figure 19).

[0528] After a 30-minute delay, 75 μL of 3.0 M sodium carbonate was added to raise the pH of the mixture to approximately 8.3. Microbial cells were centrifuged and washed using the automated fluid and centrifuge described in international patent application PCT / CA2015 / 050449, and the cells were resuspended in 1 mM phosphate buffer to obtain approximately 100 μL of microbial suspension. Each of the obtained microbial suspensions was applied to a blood agar plate and incubated overnight at 37°C, after which the colonies were counted. Results for different microorganisms are shown in Figure 25, showing a 50% excess recovery rate across all microbial species tested.

[0529] Experiments were also conducted to demonstrate the ability of hemolytic reagents and microbial cell isolation methods to yield microbial cell suspensions of suitable concentrations and purification for nucleic acid amplification. Nevertheless, there are risks associated with large volumes of whole blood in terms of increased risk of incomplete hemolysis in microbial cell suspensions and increased risk of introducing higher concentrations of RT-LAMP inhibitors.

[0530] To test for the completion of hemolysis and the potential for RT-LAMP inhibition, an 8 mL sample of SPS whole blood previously collected in a BD Vacutainer® SPS tube was combined in a separate tube with either hemolysis reagent BLR-1 or BLR-2 (2 mL) to obtain a first mixture. The total volume of whole blood used from the SPS tube in the blood / hemolysis reagent mixture was approximately 6.4 mL, and it was estimated that the SPS tube contained approximately 20% SPS solution. The tube was capped, and the blood and hemolysis reagent mixture was inverted 7-10 times to ensure proper mixing and hemolysis of the blood.

[0531] After 1 hour, the first mixture (8 mL) was mixed with 3.0 M sodium carbonate (100 μL) to obtain the second mixture, and the pH was raised above 9 to complete dissolution, producing a second mixture with lower viscosity. The 8 mL second mixture was based on approximately 5.12 mL of soluble whole blood, 1.6 mL of hemolytic reagent, and 1.28 mL of original SPS / saline solution from a BD Vacutainer® SPS tube. The cartridge was then placed in an automated fluid and centrifuge as described in international patent application PCT / CA2015 / 050449, and automated centrifugation and washing with 1 mM phosphate buffer were performed to obtain approximately 120 μL of the third mixture, which was then subjected to thermal dissolution conditions at 95°C for 10 minutes. This was then spiked with a lysate derived from Klebsiella pneumoniae to obtain a fourth mixture having a final cell lysate concentration of approximately 250 CFU / mL. 4.38 μL of the fourth mixture was then combined with a solution of Klebsiella pneumoniae-specific RT-LAMP primers (0.63 μL) and SYTO® 9 dye to obtain a fifth mixture (5 μL) consisting of the third mixture (87.5%) obtained through an automated process. This fifth mixture of lysate, dye, and primer was then used to rehydrate the dried RT-LAMP enzyme at the bottom of individual wells in a PCR plate. For control, the same lysate was spiked in 1 mM phosphate buffer (PB) and amplified using the same RT-LAMP protocol.

[0532] Figure 26 summarizes the RT-LAMP TTP data for Klebsiella pneumoniae lysate in blood matrices prepared from a total of 20 different blood donors using either BLR-1 or BLR-2. Each blood donor was tested with either BLR-1 or BLR-2 along with a control. With the third mixture derived from BLR-1, the mean TTP for Klebsiella pneumoniae lysate was 7.82 ± 0.77 minutes, or a difference of 0.82 minutes, compared to its control at 7.00 ± 0.36 minutes. The maximum time difference measured from the control to BLR-1 was less than +2.0 minutes, while the minimum time difference was -0.84 minutes. From BLR-2, the mean was 7.21 ± 0.76 minutes, compared to its control at 7.00 ± 0.39 minutes, or a difference of 0.21 ± 0.52 minutes. The maximum time difference compared to the control for BLR-1 was +0.91 minutes, while the minimum time difference was -1.10 minutes. Overall, BLR-2 resulted in lower TTP than BLR-1 (p=0.0162) and was close to the control (p=0.0029). This suggests the presence of fewer RT-LAMP inhibitors in the matrix.

[0533] We also conducted research on automated isolation of microbial cells from whole blood samples, as well as rapid detection of microbial cells by nucleic acid amplification and detection. All samples, individually spiked with either Gram-positive or Gram-negative species, were processed according to the method shown in Figure 19 to obtain the corresponding microbial cell suspensions. Each microbial cell suspension was then analyzed using an RT-LAMP assay with target-specific primers.

[0534] For each test, 8 mL of whole human blood, previously collected in a BD Vacutainer® SPS tube, was combined in the tube with either the BLR-1 or BLR-2 hemolytic reagent. The tube was capped, and the blood and reagent mixture was inverted 7–10 times to ensure proper mixing and hemolysis of the blood. After removing 8 mL of hemolytic blood, the tubes were spiked with approximately 30–50 CFU of microorganisms.

[0535] After 30 minutes to 1 hour, the mixture was mixed using an automated process in a fluid cartridge containing 3.0 M sodium carbonate (100 μL) to raise the pH to slightly above 9, completing the dissolution and producing a low-viscosity mixture. Furthermore, SE-15 defoamer (1.6 μL) was added to ensure that any foam generated in the cartridge's fluid system dissipated quickly. The cartridge was processed using an automated fluid and centrifuge as described in international patent application PCT / CA2015 / 050449, followed by automated centrifugation and washing with 1 mM phosphate buffer, and then centrifugation with 1 mM phosphate buffer to obtain approximately 100 μL of microbial cell suspension.

[0536] Next, cells in a microbial cell suspension were lysed by either heat or glass beads to release mRNA for molecular amplification by RT-LAMP. Then, 1 microliter of the lysate was combined with 4 μL of a mixture containing RT-LAMP reagent, stabilizer, STYO® 9 dye, and a set of target-specific primers. Using a LightCycler® 96 system, 4 to 6 reactants in a volume of 5 μL were amplified for each lysate, and the average TTP was measured. As a control, the same 50 CFU of cells were added to 1 mM phosphate buffer (100 μL), lysed by either heat or glass beads, and then amplified using the same RT-LAMP procedure.

[0537] Figure 27 shows the LAMP TTP for various microorganisms spiked in blood. For BLR-1, the mean time difference (Δ(TTP)) compared to the control was +1.36 ± 0.64 minutes for all tested microorganisms. However, from the aforementioned LAMP inhibition studies, the mean time delay of 0.82 minutes due to BLR-1 could be attributed to molecular inhibition of the LAMP reaction by the blood matrix. For BLR-2, the time difference was +1.21 ± 0.78 minutes, with the mean 0.21 minutes potentially attributable to molecular inhibition. Note that increasing the pH to approximately 9 was beneficial in reducing viscosity to complete blood digestion and produce a microbial cell suspension free of red debris. Although this high pH level is considered detrimental to cell viability, some loss of viability does not appear to significantly affect detection by microbial RNA in molecular assays.

[0538] The specific embodiments described above are presented as examples, and it should be understood that various modifications and alternative forms are possible. It should also be understood that the claims are not intended to limit the scope to any specific form disclosed, but rather to encompass all modifications, equivalents, and alternative forms that fall within the spirit and scope of this disclosure.

Claims

1. A method for performing nucleic acid amplification using nucleic acids derived from microcolonies of microbial cells, To detect the presence of the microcolonies in the solid growth medium, The process involves recovering microbial cells from the microcolony while its diameter remains less than 100 microns, thereby obtaining the recovered microbial cells. The recovered microbial cells are lysed to obtain a lysate, The method involves performing nucleic acid amplification in the absence of an intervening proliferation step to amplify at least one nucleic acid present in the lysate, thereby obtaining an amplification product. A method that includes this.

2. The method according to claim 1, further comprising detecting the presence of the amplification product.

3. The method according to claim 2, wherein at least one nucleic acid amplified by nucleic acid amplification includes a drug resistance gene.

4. The method according to claim 2, wherein the nucleic acid amplification is configured to amplify a plurality of nucleic acids.

5. The method according to claim 4, wherein at least one of the plurality of nucleic acids includes a drug resistance gene.

6. The method according to claim 4, wherein the plurality of nucleic acids include a set of drug resistance genes.

7. The method according to claim 4, wherein at least one of the plurality of nucleic acids includes a drug resistance gene, and at least one of the plurality of nucleic acids is uniquely associated with a microbial species.

8. The method according to claim 4, wherein at least one of the plurality of nucleic acids includes a drug resistance gene, and at least one of the plurality of nucleic acids is uniquely associated with a microbial genus.

9. The method according to claim 4, wherein at least one of the plurality of nucleic acids includes a drug resistance gene, and at least one of the plurality of nucleic acids is uniquely associated with a microbial strain.

10. The method according to claim 4, wherein at least two of the plurality of nucleic acids are associated with drug resistance genes, and at least two of the plurality of nucleic acids are associated with a specific microbial genus, strain, or species.

11. The method according to any one of claims 4 to 10, wherein at least two of the nucleic acids are amplified by a multiple amplification reaction.

12. The method according to any one of claims 1 to 11, wherein the nucleic acid amplification is carried out without performing nucleic acid extraction from the lysate.

13. The method according to any one of claims 1 to 12, wherein the microbial cells are Gram-positive microbial cells, and the lysis of the microbial cells is carried out by thermal lysis.

14. The method according to claim 1, further comprising performing sequencing on the amplification product.

15. The method according to claim 14, wherein the nucleic acid amplification includes whole genome amplification.

16. Before detecting the presence of the microcolony, Obtaining a sample, Without culturing the sample, microbial cells from the sample are seeded onto the solid growth medium. The solid growth medium is incubated, and the solid growth medium is monitored for the presence of one or more microcolonies. The method according to any one of claims 1 to 15, further comprising:

17. The method according to claim 16, wherein seeding microbial cells from the sample into the solid growth medium includes bringing the sample into direct contact with the solid growth medium.

18. The method according to claim 16, wherein the sample is a urine sample.

19. Before seeding the microbial cells from the sample onto the solid growth medium, Microbial cells are separated from the sample, and the microbial cells are resuspended to obtain a microbial suspension. The method according to claim 16, further comprising, wherein seeding the microbial cells from the sample involves contacting the microbial suspension with the solid growth medium.

20. The aforementioned sample includes whole blood, and the isolation of the microbial cells from the aforementioned sample is The aforementioned sample is brought into contact with a blood lysis reagent containing saponin and sodium polyanethole sulfonate, and a mixture is formed using an alkaline buffer. The microbial cells are separated from the mixture and the microbial cells are resuspended to obtain the microbial suspension. The method according to claim 19, including the method described in claim 19.

21. The method according to any one of claims 16 to 20, wherein the microbial cells are seeded in the solid growth medium such that the area ratio of residual sample debris is 10% to 50%.

22. The method according to any one of claims 16 to 20, wherein the microbial cells are seeded in the solid growth medium such that the area ratio of residual sample debris is 10% to 40%.

23. The method according to any one of claims 16 to 20, wherein the microbial cells are seeded in the solid growth medium such that the area ratio of residual sample debris is 10% to 30%.

24. The method according to any one of claims 16 to 20, wherein the microbial cells are seeded in the solid growth medium such that the average lateral dimension of the residual sample debris is 1 to 10 micrometers.

25. The microcolony is a first microcolony, the second microcolony is detected in the solid growth medium, and the method is After collecting the first microcolony, monitoring of the second microcolony is continued until it grows to a size suitable for performing the downstream assay. To recover the second microcolony, The downstream assay is performed using microbial cells recovered from the second microcolony. The method according to any one of claims 1 to 24, further comprising:

26. The method according to claim 25, wherein the downstream assay is selected from matrix-assisted laser desorption / ionization and antimicrobial susceptibility testing.

27. A method for performing nucleic acid amplification using nucleic acids derived from microcolonies of microbial cells, To detect the presence of microcolonies in solid growth medium, The microcolony is monitored to identify when it has grown into a colony containing sufficient biomass to facilitate sequencing. To recover microbial cells from the colony, The recovered microbial cells are lysed to obtain a lysate, In the absence of the intervening proliferation step and the intervening amplification step, the nucleic acid from the lysate is used to prepare a library for sequencing. A method that includes this.

28. The method according to claim 27, further comprising performing sequencing based on a prepared library.

29. A method for performing Gram staining on microbial cells recovered from microcolonies, To detect the presence of the microcolonies in a solid growth medium, The microbial cells are collected from the microcolony while the diameter of the microcolony remains less than 100 microns, thereby obtaining the collected microbial cells. Gram staining is performed on the recovered microbial cells in the absence of the intervening growth step. A method that includes this.

30. A method for performing nucleic acid amplification using nucleic acids derived from microcolonies of microbial cells, To detect the presence of the microcolonies in the solid growth medium, The process involves recovering material from the microcolony while the diameter of the microcolony remains less than 100 microns, In the absence of an intervening proliferation step, nucleic acid amplification is performed using nucleic acids present with the recovered material. A method that includes this.

31. A method for detecting infectious diseases, Obtaining a sample, Without culturing the aforementioned sample, microbial cells from the aforementioned sample are seeded onto a solid growth medium. The solid growth medium is incubated, and the solid growth medium is monitored for the presence of one or more microcolonies. To detect multiple microcolonies in the aforementioned solid growth medium, To identify indicators related to the severity of the infection using the number of microcolonies. A method that includes this.

32. A method for monitoring infectious diseases, a) Obtaining a sample, b) Seeding microbial cells from the sample into a solid growth medium without culturing the sample, c) Incubating the solid growth medium and monitoring the solid growth medium for the presence of one or more microcolonies, d) Detecting multiple microcolonies in the solid growth medium, e) After a time delay, repeat steps a) to d), f) Repeat step e) at least once, g) Monitoring infectious diseases using the time dependence of the number of detected microcolonies and A method that includes this.

33. A method for processing whole blood samples suspected of containing microbial cells, The method involves collecting the whole blood sample and mixing the whole blood sample with a hemolytic reagent to obtain a first mixture, wherein the hemolytic reagent comprises saponin and sodium polyanetholesulfonate. The first mixture is brought into contact with an alkaline buffer solution to obtain a second mixture having a viscosity less than that of the first mixture. The microbial cells are separated from the second mixture to obtain a suspension containing the microbial cells. A method that includes this.

34. The method according to claim 33, wherein the first mixture is brought into contact with the second mixture after a time delay of 15 minutes to 24 hours to allow for partial digestion of the blood debris in the first mixture before contact with the alkaline buffer.

35. The method according to claim 33, wherein the first mixture is brought into contact with the second mixture after a time delay of 30 minutes to 24 hours to allow for partial digestion of the blood debris in the first mixture before contact with the alkaline buffer.

36. The method according to claim 33, wherein the first mixture is brought into contact with the second mixture after a time delay of 40 minutes to 24 hours to allow for partial digestion of the blood debris in the first mixture before contact with the alkaline buffer.

37. The method according to claim 33, wherein the first mixture is brought into contact with the second mixture after a time delay of 15 minutes to 1 hour to allow for partial digestion of the blood debris in the first mixture before contact with the alkaline buffer.

38. The method according to claim 33, wherein the first mixture is brought into contact with the second mixture after a time delay of 30 minutes to 1 hour to allow for partial digestion of the blood debris in the first mixture before contact with the alkaline buffer.

39. The method according to claim 33, wherein the first mixture is brought into contact with the second mixture after a time delay of 40 minutes to 1 hour to allow for partial digestion of the blood debris in the first mixture before contact with the alkaline buffer.

40. The method according to any one of claims 34 to 39, wherein the volume of the whole blood sample is 5 ml to 10 ml.

41. To lyse the microbial cells in the suspension and obtain a lysate, The method involves performing nucleic acid amplification to amplify at least one nucleic acid present in the lysate, thereby obtaining an amplification product. The method according to any one of claims 33 to 40, further comprising:

42. The method according to claim 41, further comprising detecting the presence of the amplification product.

43. The method according to claim 41, wherein at least one nucleic acid amplified by nucleic acid amplification includes a drug resistance gene.

44. The method according to claim 41, wherein the nucleic acid amplification is configured to amplify a plurality of nucleic acids.

45. The method according to claim 44, wherein at least one of the plurality of nucleic acids includes a drug resistance gene.

46. The method according to claim 44, wherein the plurality of nucleic acids include a set of drug resistance genes.

47. The method according to claim 44, wherein at least one of the plurality of nucleic acids includes a drug resistance gene, and at least one of the plurality of nucleic acids is uniquely associated with a microbial species.

48. The method according to claim 44, wherein at least one of the plurality of nucleic acids includes a drug resistance gene, and at least one of the plurality of nucleic acids is intrinsically associated with a microbial genus.

49. The method according to claim 44, wherein at least one of the plurality of nucleic acids includes a drug resistance gene, and at least one of the plurality of nucleic acids is uniquely associated with a microbial strain.

50. The method according to claim 44, wherein at least two of the plurality of nucleic acids are each associated with a drug resistance gene, and at least two of the plurality of nucleic acids are each associated with a specific microbial genus, strain, or species.

51. The method according to any one of claims 44 to 50, wherein at least two of the nucleic acids are amplified by a multiple amplification reaction.

52. The method according to any one of claims 41 to 51, wherein the nucleic acid amplification is carried out without performing nucleic acid extraction from the lysate.

53. The method according to claim 41, further comprising performing sequencing on the amplification product.

54. The suspension of microbial cells is seeded onto a solid growth medium, The aforementioned solid growth medium is monitored for the presence of microcolonies. The method according to any one of claims 33 to 53, further comprising:

55. The method according to claim 54, further comprising detecting the presence of the microcolony.

56. The method according to claim 54, wherein the composition of the hemolytic reagent and the time delay between forming the first mixture and contacting the first mixture with the alkaline buffer are selected such that, after seeding the suspension of microbial cells onto the solid growth medium, residual blood debris from the suspension partially covers the seeding surface of the solid growth medium with a spatial area ratio of less than 50 percent.

57. The method according to claim 54, wherein the composition of the hemolytic reagent and the time delay between forming the first mixture and contacting the first mixture with the alkaline buffer are selected such that, after seeding the suspension of microbial cells onto the solid growth medium, residual blood debris from the suspension partially covers the seeding surface of the solid growth medium with a spatial area ratio of less than 40 percent.

58. The method according to claim 54, wherein the composition of the hemolytic reagent and the time delay between forming the first mixture and contacting the first mixture with the alkaline buffer are selected such that, after seeding the suspension of microbial cells onto the solid growth medium, residual blood debris from the suspension partially covers the seeding surface of the solid growth medium with a spatial area ratio of less than 30 percent.

59. The method according to claim 54, wherein the composition of the hemolytic reagent and the time delay between forming the first mixture and contacting the first mixture with the alkaline buffer are selected such that, after seeding the suspension of microbial cells onto the solid growth medium, residual blood debris from the suspension partially covers the seeding surface of the solid growth medium by a spatial area ratio of less than 20 percent.

60. The method according to claim 54, wherein the composition of the hemolytic reagent and the time delay between forming the first mixture and contacting the first mixture with the alkaline buffer are selected such that the average size of residual blood debris particles present in the suspension is less than 10 micrometers.

61. The method according to any one of claims 33 to 60, wherein the whole blood sample is collected in a vacuum container containing the hemolytic reagent.

62. The method according to any one of claims 33 to 61, wherein the microbial cells are separated from the second mixture within one hour after contacting the first mixture with the alkaline buffer.

63. The method according to any one of claims 33 to 62, wherein the hemolytic reagent is provided such that, after mixing the hemolytic reagent with the whole blood sample, the first mixture contains saponin at a concentration of 0.75 to 60 mg / ml and sodium polyanetholesulfonate at a concentration of 0.35 to 50 mg / ml.

64. The method according to any one of claims 33 to 63, wherein the pH of the blood lysis reagent is 3.5 to 8 before contact with the whole blood sample.

65. The method according to any one of claims 33 to 64, wherein the concentration of calcium ions in the blood lysis reagent relative to the dry weight of the saponin is less than 0.5% w / w.

66. The method according to any one of claims 33 to 64, wherein the concentration of calcium ions in the blood lysis reagent relative to the dry weight of the saponin is 0.1% w / w to 0.5% w / w.

67. The method according to any one of claims 33 to 64, wherein the pH of the alkaline buffer solution is 8 to 9.

68. The method according to any one of claims 33 to 67, wherein the blood lysis reagent further comprises cyclodextrin having a concentration of 0.1 mM to 20 mM.

69. The method according to any one of claims 33 to 67, wherein the blood lysis reagent further comprises cyclodextrin having a concentration of 1 to 5 mM per 3% w / w saponin.

70. The method according to any one of claims 33 to 69, wherein the whole blood sample is collected at a collection site and mixed with the hemolytic reagent, and the first mixture is received at a processing location away from the collection site before the first mixture is brought into contact with the alkaline buffer.

71. The method according to claim 70, further comprising transporting the first mixture from the collection location to the processing location.

72. The method according to any one of claims 33 to 71, wherein the alkaline buffer is mixed with the first mixture in a fluid cartridge, and an automated device is used to process the fluid cartridge to carry out the automated separation of the microbial cells.

73. The method according to any one of claims 33 to 72, wherein the second mixture is formed by contacting a portion of the first mixture with the alkaline buffer solution.

74. A kit for processing whole blood samples suspected of containing microbial cells, A container containing a hemolytic reagent, wherein the hemolytic reagent comprises saponin and sodium polyanethole sulfonate, and the container has an internal pressure low enough to facilitate the collection of 5 ml to 10 ml of the whole blood sample from a subject, thereby forming a first mixture when the whole blood sample is drawn into the container under pressure. A second container containing an alkaline buffer, wherein the alkaline buffer is configured such that, after mixing the first mixture with the alkaline buffer to form a second mixture, the viscosity of the second mixture is less than the viscosity of the first mixture. A kit that includes this.

75. The kit according to claim 74, wherein the concentration of calcium ions in the blood lysis reagent is less than 0.5% w / w.

76. The kit according to claim 74, wherein the concentration of calcium ions in the blood lysis reagent is 0.1% w / w to 0.5% w / w.

77. The kit according to any one of claims 74 to 76, wherein the blood lysis reagent further comprises cyclodextrin having a concentration of 0.1 mM to 20 mM.

78. A blood lysis collection container comprising a blood lysis reagent, wherein the blood lysis reagent comprises saponin and sodium polyanethole sulfonate, the container has sufficient pressure to facilitate the collection of 5 ml to 10 ml of whole blood sample from a subject, thereby forming a first mixture when the whole blood sample is drawn into the container, and the concentration of calcium ions in the blood lysis reagent is 0.1% to 0.5% w / w.

79. A method for recovering microcolonies from a solid growth medium, Between the solid growth medium and the conductive elongated member, a potential difference is applied such that the conductive elongated member has a positive polarity with respect to the solid growth medium. The distal end region of the conductive elongated member is brought sufficiently close to the microcolony in order to facilitate the collection of microbial cells from the microcolony via an electric field to the distal end region. A method that includes [this].

80. The method according to claim 79, wherein the distal end region includes an electrically insulating layer that prevents direct electrical contact between the conductive portion of the conductive elongated member and the collected microbial cells.

81. The method according to claim 79 or 80, wherein the potential difference is 3V to 50V.

82. A method for recovering microcolonies from a solid growth medium, To provide a fluid transfer device including a fluid transfer tube, wherein the fluid transfer tube has a distal end including an aperture suitable for fluid suction and distribution, Controlling the fluid transfer device to aspirate the recovered buffer solution, The fluid transfer tube is positioned such that its distal end brings the solid growth medium into contact with the aperture surrounding the microcolony, thereby surrounding the microcolony and forming a seal between the fluid transfer tube and the solid growth medium. The fluid transfer device is controlled to distribute at least a portion of the recovered buffer so that at least a portion of the distributed recovered buffer enters the solid growth medium, thereby releasing microbial cells from the microcolonies and forming a suspension containing the released microbial cells. Controlling the fluid transfer device to aspirate at least a portion of the suspension. A method that includes this.

83. The method according to claim 82, wherein during the distribution of the recovered buffer solution and the aspiration of the suspension, the distal end of the fluid transfer device is located below the surface of the solid growth medium.

84. The method according to claim 83, wherein the distal end of the fluid transfer device extends to a depth of 0.1 to 0.6 mm below the surface of the solid growth medium.

85. The method according to claim 83, wherein the distal end of the fluid transfer device extends to a depth of 0.1 to 0.4 mm below the surface of the solid growth medium.

86. The method according to claim 83, wherein the distal end of the fluid transfer device extends to a depth of 0.1 to 0.6 mm below the surface of the solid growth medium.

Images