Microorganism capture from antimicrobial-containing solution

Abstract

To provide a method for recovering viable microorganisms from a sample containing an antimicrobial agent.SOLUTION: A method for recovering viable microorganisms from a sample comprising microbial cells and an antimicrobial agent, the method comprises: (a) incubating the sample with coated particles to form particle-microorganism complexes; and (b) separating the particle-microorganism complexes from the antimicrobial agent, thereby recovering viable microorganisms from the sample, wherein the sample is derived from a subject treated with the antimicrobial agent, the microorganisms in the sample are susceptible or presumed to be susceptible to the antimicrobial agent, and the coated particles bind to the microorganisms through non-specific binding without using a target-specific ligand.SELECTED DRAWING: Figure 1

Description

Detailed Description of the Invention

[0001] [Technical field] The present invention generally relates to the field of recovering microorganisms from samples containing antimicrobial agents. Thus, the method of the present invention allows for the determination of the absence or presence of microbial pathogens in clinical samples, such as unpurified blood, blood cultures, and other body fluids. The present invention also relates to kits containing reagents useful for carrying out the method. Applications of the present invention include determining the presence or absence of viable microorganisms, their Gram status, species, and antimicrobial susceptibility / resistance. The present invention can also be used to monitor the effectiveness of antimicrobial treatment of patients. [Background technology]

[0002] The present inventors have recognized that in samples taken from subjects suspected of having a microbial infection, there are much higher levels of nucleated blood cells (white blood cells) than previously thought, even though the majority of the samples are not actually from infected subjects. This has created a need for improved methods of separating potential microorganisms from blood cells, particularly white blood cells, in blood samples taken from patients being screened for infection.

[0003] When blood samples are taken from patients who have already received antibiotics, subsequent blood cultures often do not grow microorganisms, even when the patient is clearly infected (Scerbo et al., Surg Infect (Larchmt). 2016 June; 17(3):294-302; Sinha et al., Clin Microbiol Rev. 2018 February 28; 31(2):e00089-17; Scheer et al., Clin Microbiol Infect. 2019 March; 25(3):326-331).

[0004] To address this challenge, blood culture suppliers have offered blood culture bottles containing antibiotic-absorbent resins (e.g., BACTEC PLUS bottles from Becton Dickinson and BacT / Alert bottles from bioMérieux). Flayhart et al. (J. Clin. Microbiol. (2007), pp. 816-821) describe testing of BACTEC PLUS and BacT / Alert bottles under simulated conditions. In the presence of ceftriaxone, neither system was able to recover Streptococcus pneumoniae.

[0005] Chung et al. (Eur J Clin Microbiol Infect Dis. (2019), 38(12):2229-2233) also describe testing BacT / Alert and BACTEC Plus bottles under simulated conditions. Both systems demonstrated low to zero detection rates in the presence of certain antimicrobial agents. For example, E. coli or K. pneumoniae in the presence of cefepime; E. coli in the presence of cefotaxime; and E. coli, K. pneumoniae, or P. aeruginosa in the presence of meropenem. Thus, neither system can universally recover microorganisms from samples containing antimicrobial agents.

[0006] In WO 2009 / 007719, NAD-dependent ligases are described as useful indicators of the presence of microorganisms in a sample. Ligases are enzymes that catalyze the joining of nucleic acid molecules. The ligation reaction requires either ATP or NAD+ as a cofactor, depending on the ligase involved.

[0007] WO 2011 / 130584 describes a method for detecting viable microorganisms based on the detection of DNA or RNA polymerase, in which a sample is contacted with a nucleic acid substrate that acts as a substrate for the microbial polymerase, incubated under conditions suitable for polymerase activity from intact microorganisms, and any resulting nucleic acid products are determined using a nucleic acid amplification technique such as quantitative polymerase chain reaction. Such assays are called "ETGA assays," where ETGA stands for Enzymatic Template Generation and Amplification. A problem with ETGA assays for viable microorganisms in crude samples is the presence of contaminating polymerase activity, separate from the microorganisms, originating from host (e.g., human) cells and dead microorganisms. The ETGA assay cannot distinguish microbial polymerase activity from that of the host or dead microorganisms.

[0008] WO 2010 / 119270 describes a method for removing DNA ligase activity apart from intact microorganisms. WO 2011 / 070507 describes the selective lysis of animal cells using a non-ionic detergent and a buffer. WO 2017 / 182775 describes a method for detecting the absence or presence of microorganisms in a sample that may also contain non-microbial cells, comprising selectively lysing non-microbial cells, filtering the lysate, and detecting the absence or presence of microorganisms retained in or on the filter.

[0009] The use of magnetic beads coated with specific binding moieties such as antibodies is known for the capture of target species. The specificity of these products is defined by the specificity of the antibody or other binding ligand, which is generally chosen for the specific purpose of being highly specific to allow the isolation of a particular microorganism.

[0010] WO 03 / 102184 describes methods, compositions, and kits for concentrating or separating cells (e.g., bacteria) using flocculating agents, such as polyamines or cationic surfactants, to form complexes with the cells that flocculate them. Separation of the flocculated cells can be achieved using a solid phase capable of binding to the cells, such as magnetic beads.

[0011] WO 01 / 53525 describes a method for isolating cells (e.g., microorganisms) from a sample, which method involves binding the cells to a solid support by means of a carbohydrate ligand immobilized on the solid support. A kit for carrying out such a method is sold by DiaSorin Molecular ("Bugs'n Beads™" kit).

[0012] Other kits for isolating microorganisms include ApoH-Technologies Peps6 magnetic beads, which are coated with Peps6, a synthetic molecule derived from apolipoprotein H protein (ApoH), also known as beta-2 glycoprotein.

[0013] Cartwright et al. (EBioMedicine (2016) 9:217-227) describe the use of magnetic beads coated with mannose-binding lectin to capture pathogen-associated molecular patterns (PAMPs). PAMPs, according to Cartwright, are carbohydrate cell wall material and outer membrane vesicles released by various types of live and dead pathogens. Cartwright notes that the release of PAMPs by pathogens increases when they are killed by antibiotics. The Cartwright assay is not intended to recover viable microorganisms from a sample.

[0014] WO 2018 / 044966, EP 1118676, CN 109929763, CN 109741896 describe the use of beads to capture microorganisms from samples that do not contain antimicrobial agents. [Description of the Invention]

[0015] The present invention relates to the recovery of viable microorganisms from a sample containing microbial cells and an antimicrobial agent by capturing the microorganisms with coated particles to form a microbial complex and separating the microbial complex from the antimicrobial agent. The inventors have discovered that coated particles can universally bind to microorganisms, thus allowing the recovery of a range of microorganism types, such as bacteria and fungi, without prior knowledge of which types are present in the sample. Pan-microbial recovery according to the present invention is effective in eliminating the growth-inhibitory effects of antimicrobial agents. This is an alternative and provides a more effective way to address challenges currently addressed in clinical settings by using blood culture bottles containing antibiotic-absorbent resins, as demonstrated in comparative experiments reported herein. As demonstrated herein, recovered microorganisms remain viable and rapidly recover to demonstrate growth. The inventors surprisingly found that this recovery can be achieved with coated particles lacking complex moieties, such as antibodies, that bind to (specific) microorganisms in a targeted manner.

[0016] In addition to rescuing microorganisms from antimicrobial agents, the present invention also allows for the rescue of microorganisms from inhibitors of subsequent analysis, such as blood cell remnants, hemoglobin, and leukocyte DNA, which can have significant impacts on techniques such as molecular detection and identification. Platelet removal may also be useful in some techniques for measuring antimicrobial susceptibility.

[0017] The present invention also provides significant concentrations of microorganisms (e.g., microorganisms in a 5 mL blood sample can be concentrated to tens of μL), particularly when compared to blood culture bottles containing antibiotic-absorbent resins (e.g., BACTEC PLUS bottles from Becton Dickinson and BacT / Alert from bioMérieux). This is advantageous for downstream applications requiring higher concentrations of microorganisms. Various tests, including molecular tests, can be performed to detect and / or characterize the recovered viable microorganisms, and a more concentrated sample is generally necessary for or advantageous in the context of such assays. Suitable assays are discussed herein, including EGTA assays, PCR or sequencing assays, and antimicrobial susceptibility testing (AST), particularly multi-well AST, which are performed in relatively low volumes. Providing an appropriately concentrated sample initially improves processing time and may avoid further concentration steps in downstream microbial testing.

[0018] method The present invention provides a method for recovering viable microorganisms from a sample containing microbial cells and an antimicrobial agent, the method comprising the steps of: a) incubating the sample with coated particles to form particle-microorganism complexes; and b) separating the particle-microorganism complexes from the antimicrobial agent, thereby recovering viable microorganisms from the sample.

[0019] The term "viable microorganisms" refers to non-dead microorganisms and applies to all aspects of the disclosure. Thus, they are microorganisms that have not been killed by an antimicrobial agent. Viable microorganisms may be capable of metabolic recovery from exposure to an antimicrobial agent and / or growth under appropriate conditions (once recovered from a sample containing an antimicrobial agent, as described herein). "Growth" refers to both an increase in volume / size and the ability to grow, particularly the ability to proliferate (through synthesis, DNA replication, and cell division).

[0020] When viable microorganisms are recovered from a sample, the concentration of antimicrobial agent to which the recovered viable microorganisms are exposed can be substantially reduced. When viable microorganisms are recovered from a sample, the concentration of viable microorganisms can be increased relative to the sample. This increased concentration of viable microorganisms can aid in downstream processes, such as characterization of the viable microorganisms.

[0021] The step of "incubating the sample" refers to contacting the sample with the coated particles under conditions conducive to the formation of particle-microorganism complexes. In some embodiments, the step of incubating the sample with the coated particles includes contacting the coated particles with the sample at a predetermined temperature (e.g., 37°C) for a period of time (e.g., 30 minutes). Incubation can be performed with or without shaking (e.g., with a platform shaker, orbital shaker, or shaking incubator set at 500-1000 rpm). Incubation can be performed in the absence of a fixative. The fixative can be a cross-linking fixative or a non-cross-linking fixative. Cross-linking fixatives function by forming chemical bonds between microorganisms in the sample. Non-cross-linking fixatives do not chemically alter the microorganisms in the sample, but rather simply precipitate them.

[0022] The method can further include the step of incubating and / or culturing the recovered viable microorganisms. The viable microorganisms can be incubated and / or cultured while attached to the coated particles (i.e., in a complex).

[0023] According to the method of the present invention, culturing the recovered viable microorganisms may include increasing the number of viable microorganisms. In this method, incubating the recovered viable microorganisms may be considered a separate phase and therefore may not include increasing the number of viable microorganisms. Microorganisms exposed to an antimicrobial agent may not be in a position to grow for a period after recovery. Therefore, incubating the recovered viable microorganisms may include allowing the organisms to undergo metabolic recovery after exposure to an antimicrobial agent. Some downstream characterization methods do not require microorganisms to grow, but metabolic recovery may be important. Therefore, some methods may not involve a culturing step after recovery. However, after metabolic recovery, the viable microorganisms may enter a growth phase. Therefore, the method of the present invention may include both incubation and culturing, allowing metabolic recovery after growth.

[0024] Thus, the present invention provides a method for incubating and / or culturing viable microorganisms recovered from a sample containing microbial cells and an antimicrobial agent, the method comprising the steps of: a) incubating the sample with coated particles to form particle-microorganism complexes; b) separating the particle-microorganism complexes from the antimicrobial agent, thereby recovering viable microorganisms from the sample; and c) incubating and / or culturing the recovered viable microorganisms.

[0025] The methods of the present invention can further include detecting and / or characterizing the recovered viable microorganisms. "Detecting" refers to determining or confirming, by any appropriate means, whether viable microorganisms are actually present in the recovered sample. "Characterizing" goes beyond detecting the absence or presence of a microorganism and provides additional information about the recovered viable microorganisms. Characterization can include determining whether the sample contains bacteria and / or fungi. This is particularly important in clinical samples, especially those collected from subjects suspected of infection (e.g., patients with possible sepsis). Characterization can additionally (i.e., first determine whether a microorganism (such as a bacterium or fungus) is present, and if so, further characterization steps can be performed) or alternatively include identifying the genus or species of the microorganism recovered from the sample. Characterization can include determining the Gram status of the microorganism (i.e., whether the bacterium is Gram-negative or Gram-positive). These types of characterization can be particularly useful for determining which type of antimicrobial agent is most appropriate for the subject from whom the sample was collected. Characterization may include determining the antimicrobial susceptibility and / or resistance of the microorganism. Any suitable method may be employed for these further steps, examples of which are discussed further herein.

[0026] Thus, the present invention provides a method for detecting the absence or presence of viable microorganisms in a sample containing an antimicrobial agent and suspected of containing a microorganism, the method comprising the steps of: a) incubating the sample with coated particles to form particle-microorganism complexes (if microorganisms are present in the sample, and if complexes cannot form); b) separating the particle-microorganism complexes from the antimicrobial agent, thereby recovering viable microorganisms from the sample; and c) detecting the absence or presence of viable microorganisms.

[0027] Similarly, the present invention provides a method for detecting the absence or presence of viable microorganisms in a sample suspected of containing a microorganism and comprising an antimicrobial agent, the method comprising the steps of: a) incubating the sample with coated particles to form particle-microorganism complexes (if microorganisms are present in the sample, and if complexes cannot form); b) separating the particle-microorganism complexes from the antimicrobial agent, thereby recovering viable microorganisms from the sample; c) incubating and / or culturing the recovered viable microorganisms; and d) detecting the absence or presence of viable microorganisms.

[0028] The present invention provides a method for detecting the absence or presence of a viable microbial infection in a subject, comprising performing any of the methods described herein on a sample from the subject. Typically, the subject has been treated with an antimicrobial agent. This is then the source of the antimicrobial agent in the sample. The sample is typically a clinical sample as discussed herein. The subject is typically a human subject, often a human subject suspected of suffering from an infection, which may be a bacterial or fungal infection.

[0029] In these methods, the method can further include characterizing the microorganism causing the infection. As discussed herein (hereinafter the discussion applies mutatis mutandis), any suitable characterization can be employed.

[0030] As already mentioned, the present invention is particularly applicable to clinical samples. Such samples typically contain non-microbial cells. In fact, they may contain primarily non-microbial cells (i.e., there are fewer, typically significantly fewer, microbial cells than non-microbial cells in the sample). Therefore, the inventor's discovery that coated particles can remove microorganisms from samples containing both antimicrobial agents and non-microbial cells is particularly advantageous in the present invention.

[0031] Thus, the present invention provides a method for recovering viable microorganisms from a sample containing microbial cells, non-microbial cells, and an antimicrobial agent, the method comprising the steps of: a) incubating the sample with coated particles to form particle-microorganism complexes; and b) separating the particle-microorganism complexes from the antimicrobial agent and non-microbial cells, thereby recovering viable microorganisms from the sample.

[0032] In these methods, the sample contains non-microbial cells, and step b) separates the particle-microorganism complexes from the antimicrobial agent and the non-microbial cells. The non-microbial cells may include blood cells. The blood cells may include red blood cells (erythrocytes) and / or white blood cells (leukocytes). For the avoidance of doubt, the non-microbial cells may be lysed before separation occurs. Thus, separation of particle-microorganism complexes from non-microbial cells encompasses separation of particle-microorganism complexes from non-microbial cell lysates. However, separation can also be performed when the non-microbial cells remain intact.

[0033] Exposure of microorganisms to antimicrobial agents is intended to kill or prevent the growth of microorganisms, which poses challenges when recovering viable microorganisms. Therefore, the method can rely on conditions intended to preserve viable microorganisms remaining in the sample after exposure to the antimicrobial agent. These microorganisms may be more susceptible to cell death and lysis. Therefore, certain agents that may be useful for preferentially lysing non-microbial cells over microbial cells in a sample may not be appropriate in certain embodiments of the present invention. Such agents include detergents, particularly at high concentrations. Therefore, the method (step b) of the present invention can be performed in the absence of detergents.

[0034] In some embodiments, step b) can be carried out in the presence of sodium polyanethole sulfonate. As demonstrated herein, this reagent is useful for the recovery of microorganisms from samples containing non-microbial cells.

[0035] In a further embodiment, step b) can additionally or alternatively be carried out in the presence of a reagent (e.g., a detergent) that selectively lyses non-microbial cells in the sample while preserving intact microorganisms present in the sample. Suitable reagents are known in the art and are further discussed herein.

[0036] In view of the increased susceptibility of microorganisms exposed to antimicrobial agents to lysis, in some embodiments, lysis of non-microbial cells is not performed to facilitate separation of microorganisms from non-microbial cells in a sample. Because coated particles preferentially bind microorganisms, there is no absolute requirement to lyse non-microbial cells in a sample. However, it may be advantageous to perform one or more wash steps to remove non-microbial cells (typically intact non-microbial cells) and / or cell lysates from the particle-microorganism complexes. Thus, step b) of the methods of the present invention may include washing the separated particle-microorganism complexes. Any suitable wash solution may be employed, and examples are discussed herein. In some embodiments, the separated particle-microorganism complexes are washed with a detergent-free solution. In some embodiments, the wash buffer comprises Tris and / or sodium chloride. In some embodiments, the wash buffer has a pH of about 7-9, e.g., 7.5-8.5. In some embodiments, the wash buffer contains a salt, such as a metal halide salt. A preferred example is sodium chloride. The salt concentration may be about 50-150 mM. Preferably, the wash buffer comprises phosphate buffered saline (PBS), Tris or Tricine.

[0037] In another embodiment, the separated particle-microorganism complexes can be washed with a solution containing a surfactant. This solution can be a weak surfactant solution to reduce the risk of lysing the microorganisms. The solution can contain a surfactant at a concentration that does not substantially lyse the microorganisms. A suitable example is polyethylene glycol sorbitan monolaurate (Tween 20), for example, 1-5% w / v, preferably about 1%. The reagent can include, for example, 1-5% w / v, preferably about 1% saponin. The microorganisms can be exposed to the surfactant solution for a period that does not substantially lyse the microorganisms. The solution can selectively lyse at least a portion of the non-microorganisms in the sample.

[0038] The reagent that selectively lyses non-microbial cells in a sample while preserving intact microorganisms present in the sample can include one or more enzymes. The one or more enzymes can include proteinases and / or DNAases. Suitable enzymes are discussed herein.

[0039] However, as already mentioned, when dealing with microorganisms that may be vulnerable after exposure to antimicrobial agents, it is preferable that any washing performed in steps a) and b) be performed in the absence of a detergent. The entire method can be performed in some embodiments in the absence of a detergent. In other embodiments, the method can be performed in the absence of a detergent, except for detecting and / or characterizing the recovered viable microorganisms. In this embodiment, the contents of the microbial cells may need to be released for analysis. A suitable lysis reagent can include a detergent. Such a detergent can be at a high concentration to ensure complete lysis of the microorganisms.

[0040] In this method, step b) typically involves some form of physical separation of the particle-microorganism complexes from the remainder of the sample. This may include the use of any suitable separation means. For example, separation may be achieved using a magnetic field. The use of a magnetic field requires that the particles are magnetic and attract the particle-microorganism complexes. Alternatively, other separation methods, such as centrifugation or filtration, may be employed. Step (b) may include or further include removing non-microbial cells from the particle-microorganism complexes by aspiration.

[0041] For the avoidance of doubt, the physical separation step or steps may be followed by a washing step. Thus, the method may further comprise (as part of step b) washing the separated particle-microorganism complexes to remove antimicrobial agent and / or non-microbial cells or lysates from the particle-microorganism complexes.

[0042] More generally, step b) can further comprise removing non-microbial cells from the particle-microorganism complexes.

[0043] If selective lysis is employed, it can be performed at any appropriate stage of the method to remove non-microbial cells from the sample (and retain viable microorganisms). Thus, it can be performed as a first processing step before incubating the sample with the coated particles. Thus, in some embodiments, prior to step a), non-microbial cells in the sample can be selectively lysed while retaining intact microorganisms present in the sample.

[0044] Selective lysis of non-microbial cells in a sample while preserving intact microorganisms present in the sample can involve osmotic lysis or the addition of a detergent. The detergent can be a weak detergent that causes lysis of non-microbial cells (e.g., blood cells) without substantially lysing microorganisms. Suitable examples include polyethylene glycol sorbitan monolaurate (Tween 20), for example, at 1-5% w / v, preferably about 1%. The reagent can also include, for example, 1-5% w / v, preferably about 1%, of saponin.

[0045] In this method, step a) is typically carried out in an aqueous solution. Step a) can be carried out in the presence of a buffer solution. The buffer solution can include any suitable buffering agent, including, but not limited to, phosphate buffered saline (PBS), TAPS ([Tris(hydroxymethyl)methylamino]propanesulfonic acid), Bicine (2-(bis(2-hydroxyethyl)amino)acetic acid), Tris (Tris(hydroxymethyl)aminomethane), Tricine (3-[N-Tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid), TAPSO (3-[N-Tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid), HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), TES (2-[[1,3-dihydroxyethyl]amino]-2-hydroxypropanesulfonic acid), PEG (PEG-1000), PEG-1200, PEG-1400, PEG-1600, PEG-1800, PEG-1900, PEG-2000, PEG-2100, PEG-2200, PEG-2300, PEG-2400, PEG-2500, PEG-2600, PEG-2700, PEG-2800, PEG-2900, PEG-3000, PEG-3100, PEG-3200, PEG-3300, PEG-3400, PEG-3500, PEG-3600, PEG-3700, PEG-3800, PEG-4000, PEG-4500, PEG-4600, PEG-4700, PEG-4800, PEG-4900, PEG-4900, PEG-4900, PEG-4900, PEG-4900, PEG-4900, Examples of suitable buffers include bis(2-(hydroxymethyl)propan-2-yl)amino)ethanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid), PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid)), cacodylate (dimethylarsinic acid), MES (2-(N-morpholino)ethanesulfonic acid), Bis-tris(2-bis(2-hydroxyethyl)amino)-2-(hydroxymethyl)propane-1,3-diol), maleate, phosphate, glycine, citrate, glycylglycine, formate, succinate, acetate, propionate, piperazine, histidine, ethanolamine, imidazole, borate, and carbonate. The buffer may have a pH of 7.4 to 8.5. Preferably, the buffer contains phosphate-buffered saline (PBS), Tris, or Tricine. In this method, step a) can be carried out in the presence of sodium chloride. The sodium chloride can be present at a concentration of 50 to 500 mM. Preferably, the sodium chloride can be present at a concentration of about 150 mM.

[0046] In this method, the reagent that selectively dissolves non-microbial cells in the sample while retaining intact microorganisms present in the sample can be a surfactant, particularly a weak surfactant.In this method, the surfactant can be non-ionic.In this method, the surfactant may not be conjugated to particles that can form complexes with microorganisms.Therefore, typically, the surfactant forms part of the solution to which the particles are added, but does not form part of the particles themselves.

[0047] As already introduced, the methods of the present invention can involve detecting and / or characterizing the recovered viable microorganisms. In some embodiments, detecting the absence or presence of a microorganism can include (i) detecting an enzymatic activity of the microorganism, (ii) detecting a nucleic acid or polypeptide from the microorganism, (iii) directly detecting the microorganism by cytometry or microscopy, or (iv) detecting the microorganism after cell culture.

[0048] Detection of the absence or presence of a microorganism in a particle-microorganism complex according to all relevant aspects of the invention can be performed according to any desired method. The method can involve detecting the simple absence or presence of one or more microorganisms. If present, it may involve quantification of the microorganism. It can also, in some embodiments, involve characterization of the nature of the microorganism. Thus, detection of bacteria and / or fungi can be performed. Differentiation of gram-positive versus gram-negative bacteria can also be performed. Identification of the species and antimicrobial susceptibility and / or resistance of the organism can also be performed.

[0049] Detection and / or characterization may occur after removal (or recovery) of the microorganisms from the particle-microorganism complex. The recovered microorganisms may be lysed prior to detection. Recovery may be of intact microorganisms or a lysate following lysis of the microorganisms (discussed in more detail herein).

[0050] Preferably, detection is performed without prior removal (or recovery) of the microorganisms from the particle-microorganism complexes. This embodiment is particularly useful when applying the invention to magnetic bead processing devices (i.e., represents a particularly useful implementation of the method of the invention when the particles are magnetic).

[0051] Detection of nucleic acid molecules associated with microorganisms is known in the art and can be performed at the DNA or RNA level. It can be performed by any suitable method, such as amplification (e.g., PCR) or sequencing (especially next-generation sequencing). Such methods can take advantage of sequence differences between microorganisms and non-microorganisms, such as human DNA and RNA. Such methods can involve lysing microorganisms (e.g., present in the form of particle-microorganism complexes) to release nucleic acid components.

[0052] Direct detection of microorganisms is also known. This can involve cytometric analysis, for example, by flow cytometry. It can involve the use of a microscope, for example, to visualize microorganisms recovered from or within particle-microorganism complexes.

[0053] Microbial detection can also be performed after cell culture to expand the number of microorganisms. Thus, the microorganisms initially captured within the particle-microorganism complexes can be cultured for a period of time before detection. Culture methods can directly detect microorganisms in the original sample. Alternatively, the microorganisms can be incubated for a shorter period (than culture) to allow metabolic recovery from exposure to the antimicrobial agent before detection (but without expansion).

[0054] However, in a preferred embodiment, detecting the absence or presence of the recovered microorganism may involve detecting an enzymatic activity associated with the microorganism. Suitable enzymatic activities are typically nucleic acid modifying activities, which are discussed in more detail herein.

[0055] Therefore, in this method, the step of detecting a microorganism can include the steps of (i) lysing the microorganism into a particle-microorganism complex; (ii) incubating the lysate with a nucleic acid molecule that acts as a substrate for the nucleic acid-modifying activity of the microorganism; and (iii) specifically determining the absence or presence of a modified nucleic acid molecule resulting from the action of the nucleic acid-modifying enzyme on the substrate nucleic acid molecule to indicate the absence or presence of the microorganism. Suitable substrates are discussed in more detail herein. "Substrate" refers to a nucleic acid molecule that is acted upon by an enzyme derived from a microorganism. Typically, such nucleic acid molecules are oligonucleotide substrates. They are synthetic nucleic acid molecules.

[0056] Lysis of the microorganisms in the particle-microorganism complex allows for the detection of nucleic acid molecules or enzymes, such as nucleic acid-modifying enzymes, within the microorganisms. Lysis can be achieved by the addition of a lysis mixture. Lysis mixtures are generally useful in the methods of the present invention. The lysis mixture can contain a specific mixture of components to ensure efficient lysis of the microorganisms without adversely affecting intracellular nucleic acid molecules and / or enzyme activity, such as nucleic acid-modifying activity. Components can be selected from carriers / serum proteins, e.g., BSA, surfactants / detergents, metal halide salts, buffers, chelating agents, etc. In its basic form, the lysis mixture of the present invention can include the following components: 1. Surfactants / Detergents 2. Serum proteins such as albumin (e.g., BSA) 3.Buffer 4. Nucleotides such as dNTPs 5. Nucleic acid molecules (which act as substrates in the assays of the invention).

[0057] A suitable lysis mixture is described below: L1: 252 mL in 360 mL LM 1.46% (w / v) BSA 0.15% Triton X100 0.15% Tween 20 L2: 36 mL in 360 mL of LM 100mM ammonium sulfate 20mM magnesium sulfate heptahydrate 100mM potassium chloride 200mM Tris-HCl [pH 8.0] L3: 36 mL in 360 mL LM 0.1 μM PTO-AS oligo 0.1 μM PTO-S1 oligo 20 mM Tris-HCl [pH 8.5] 10mM potassium chloride 10 μM EDTA 10 mM dNTP: 3.6 mL in 360 mL LM PTO-IPC stock: approximately 180 μL in 360 mL of LM H2O: Approximately 32.4 mL in 360 mL of LM

[0058] "PTO-AS oligo" refers to an antisense oligonucleotide containing phosphorothioate nucleotides. "PTO-S1 oligo" refers to a sense oligonucleotide containing phosphorothioate nucleotides. The two oligonucleotides hybridize to each other to form a substrate nucleic acid molecule.

[0059] "PTO-IPC" refers to an IPC molecule that contains phosphorothioate nucleotides.

[0060] Suitable substrates and IPC molecules are discussed in further detail herein.

[0061] Exemplary amounts and concentrations of each component are listed, but can be modified as would be readily apparent to one skilled in the art.

[0062] Lysis may also require cell disruption. For example, cells may be disrupted using a lysis mixture in combination with physical and / or enzymatic means. However, methods of lysing cells typically avoid the use of physical disruption. In some embodiments, physical disruption uses a disruptor. The disruptor can incorporate beads, such as glass beads, to lyse the cells. Suitable devices are commercially available and include the Disruptor Genie manufactured by Scientific Industries, Inc. Sonication can be utilized, for example, by applying an ultrasonic horn. Enzymatic disruption, in some embodiments, can require the use of one or more agents selected from lysostaphin, lysozyme, and / or lyticase.

[0063] Once the microorganisms, if present in the sample, are lysed, the released nucleic acids and / or enzymes can be detected to indicate whether viable microorganisms are present in (and recovered from) the sample. In some embodiments, the lysate is incubated with a nucleic acid molecule that acts as a substrate for the (microorganism's) nucleic acid-modifying activity. The absence or presence of the modified nucleic acid molecule resulting from the action of the nucleic acid-modifying enzyme on the substrate nucleic acid molecule is then determined to indicate the absence or presence of the microorganism. The nucleic acid substrate molecule is designed according to the nucleic acid-modifying activity to be detected. One of skill in the art can design an appropriate substrate nucleic acid molecule. While the initial sample may contain a non-microbial source of nucleic acid-modifying activity, the methods of the present invention prevent this contaminating activity from acting on the substrate nucleic acid molecule.

[0064] According to the methods of the present invention, exemplary nucleic acid modifying activities that can be detected include polymerase and / or ligase activity. The nucleic acid modifying enzyme can include DNA or RNA polymerase. In preferred embodiments, the DNA polymerase is a DNA-dependent DNA polymerase. In some embodiments, the DNA polymerase is DNA polymerase I. The nucleic acid modifying enzyme can include a ligase, and optionally, the nucleic acid modifying enzyme is an NAD-dependent ligase (to indicate the presence of bacteria) and / or an ATP-dependent ligase (to indicate the presence of fungi or bacteria). NAD-dependent ligase is found only in (eu)bacteria, and detecting such activity may provide an additional level of specificity. This is further discussed in WO 2009 / 007719 and WO 2010 / 119270 (the relevant disclosures of which are incorporated herein). Other nucleic acid modifying activities associated with viability, such as phosphatase, kinase, and / or nuclease activity, can alternatively be measured.

[0065] As previously discussed, the method can include washing the separated particle-microorganism complexes to remove non-microbial cells or lysates. The washing step can remove inhibitors of subsequent analysis, such as PCR inhibitors. The washing step is typically performed under conditions that do not dissociate the particle-microorganism complexes.

[0066] In some embodiments, the action of a nucleic acid modifying activity on a substrate nucleic acid molecule produces an extended nucleic acid molecule. This can be by chain extension (polymerase activity) and / or by joining two nucleic acid molecules (ligase activity). In some embodiments, a substrate that can be acted upon by either a polymerase or a ligase is utilized, as either activity indicates the presence of a microorganism in a sample. In some embodiments, related activities can be distinguished with respect to the new nucleic acid molecule that is produced.

[0067] The substrate can be a template for a nucleic acid modifying activity of a microorganism. For example, the substrate can be a template for a DNA or RNA polymerase, preferably a DNA-dependent DNA polymerase, optionally where the DNA polymerase is DNA polymerase I.

[0068] Suitable nucleic acid molecules that act as substrates for microbial nucleic acid modifying activities are described in WO 2011 / 130584, WO 2010 / 119270, and WO 2009 / 007719 (the relevant disclosures of which are incorporated herein by reference). For phosphatase activity, suitable nucleic acid molecules are disclosed in WO 2006 / 123154, the disclosure of which is incorporated herein by reference.

[0069] In a specific embodiment, the (substrate) nucleic acid molecule used in the method of the invention is at least partially double-stranded and comprises a uracil residue in the complementary strand, and the step of specifically determining the absence or presence of the modified nucleic acid molecule comprises adding uracil DNA glycosylase (UDG) to the sample to degrade the uracil residue in the complementary strand.

[0070] In certain embodiments, the (substrate) nucleic acid molecule comprises or is a DNA molecule, typically a DNA oligonucleotide. In certain embodiments, the (substrate) nucleic acid molecule comprises DNA and is partially double-stranded.

[0071] In some embodiments, the (substrate) nucleic acid molecule comprises a nucleic acid consisting of a sense oligonucleotide (DNA) strand and an antisense oligonucleotide (DNA) strand, where these two strands overlap to form a double-stranded region, and the single-stranded portion of the antisense oligonucleotide strand acts as a template, with the sense oligonucleotide strand of the double-stranded region acting as a primer to generate extension products in the presence of polymerase activity.

[0072] In certain embodiments, the first strand of the partially double-stranded (substrate) nucleic acid molecule comprises (or consists of) synthetic nucleotides (e.g., phosphorothioate nucleotides), and the second (complementary) strand comprises (or consists of) uracil residues and, optionally, synthetic nucleotides (e.g., phosphorothioate nucleotides). Preferably, the double-stranded region encompasses the 3'-terminal regions of the first and second (complementary) strands. In some embodiments, the second (complementary) strand comprises a base (e.g., dideoxycytidine) at its 3'-end that blocks DNA polymerase-mediated extension of the second strand. Such partially double-stranded (substrate) nucleic acid molecules are described, for example, in Zweitzig et al., 2012 (Characterization of a novel DNA polymerase activity assay enabling sensitive, quantitative, and universal detection of viable microbes. Nucleic Acids Research, Vol. 40, No. 14, e109, pp. 1-12, incorporated herein by reference). Preferably, the double-stranded region is at least 5 nucleotides, at least 10 nucleotides, at least 15 nucleotides, at least 20 nucleotides, or at least 25 nucleotides; optionally, the double-stranded region is 50 nucleotides or less. The first strand can be extended during an incubation step using unprotected (or standard) dNTPs by the polymerase activity of the microorganisms in the sample, as described herein, to form an extended first strand containing unprotected (or standard) nucleotides. This step relies on using the second strand as a template (upstream of the region of complementarity between the first and second strands). After the incubation step, the second (complementary) strand can be degraded by adding uracil DNA glycosylase (UDG) to the sample, leaving the extended first strand as a single-stranded molecule containing synthetic and unprotected nucleotides. Following degradation of the second strand, the extended first strand of the (substrate) nucleic acid molecule can be detected in an amplification step. The present inventors have found that the use of the partially double-stranded (substrate) nucleic acid molecules described above improves the detection of microorganisms in a sample.

[0073] In some embodiments, the substrate nucleic acid molecule is pre-modified to protect it from nuclease activity, i.e., the nucleic acid molecule is modified to protect it from nuclease activity before being added to the assay. The inventors have determined that protecting the substrate nucleic acid molecule from nuclease activity is advantageous in the context of the assays of the present invention. More specifically, the incorporation of protected nucleic acid molecules into the methods of the present invention improves the sensitivity of detection. Any suitable means can be employed to protect the nucleic acid molecule from nuclease activity. Non-limiting examples include the incorporation of methylation into the nucleic acid molecule, terminal modifications such as 3'- and / or 5'-end protection, and the incorporation of synthetic nucleotides. In specific embodiments, the synthetic nucleotide comprises a phosphorothioate nucleotide and / or a locked nucleic acid nucleotide. Preferably, the synthetic nucleotide is a phosphorothioate nucleotide. In certain embodiments, the synthetic nucleotide replaces at least one to all of the nucleotides in the nucleic acid molecule.

[0074] (Substrate) nucleic acid molecules can include any naturally occurring nucleic acids and natural or synthetic analogs that can act by nucleic acid modifying activity to generate (novel, detectable) nucleic acid molecules. Substrates can, in specific embodiments, be extended and / or ligated. In some embodiments, a combination of nucleic acid substrate molecules can be employed to allow for detection of polymerase and ligase activity.

[0075] The nucleic acid substrate may be present in excess, particularly in large molar excess, relative to the nucleic acid modifying activity (provided by the microorganism) in the sample. Since a novel extended or ligated nucleic acid molecule is to be detected, only the presence of this molecule in the sample is essential for the detection method to work effectively. Therefore, the presence of other nucleic acid molecules in the sample, such as those derived from the microorganism to be detected or from mammals or other sources that may be found in the sample to be tested, is not detrimental to the method of the present invention.

[0076] The inventors have previously investigated the use of internal positive control (IPC) molecules in the context of these methods. Thus, in all aspects, the present invention may rely on the inclusion of an IPC molecule. In some embodiments, the IPC is included with the substrate nucleic acid molecule so that the IPC is exposed to identical conditions. In some embodiments, the IPC molecule is pre-modified to protect it from nuclease activity, i.e., the nucleic acid molecule is modified to protect it from nuclease activity before being added to the assay. The inventors have determined that protecting the IPC molecule from nuclease activity is advantageous in the context of the assays of the present invention. Any suitable means can be employed to protect the nucleic acid molecule from nuclease activity. Non-limiting examples include the incorporation of methylation into the nucleic acid molecule, terminal modifications such as 3'- and / or 5'-end protection, and the incorporation of synthetic nucleotides. In specific embodiments, the synthetic nucleotides include phosphorothioate nucleotides and / or locked nucleic acid nucleotides. Preferably, the synthetic nucleotides are phosphorothioate nucleotides. In certain embodiments, synthetic nucleotides replace at least one and up to all of the nucleotides in the IPC molecule. Preferably, the substrate and the IPC molecule are modified in the same way, which is advantageous so that they behave similarly in the assays of the invention.

[0077] In some embodiments, an internal positive control (IPC) nucleic acid molecule contains identical primer binding sites to a substrate nucleic acid molecule such that there is competition for primer binding in a nucleic acid amplification reaction that includes both the nucleic acid molecule and the IPC.

[0078] In all methods of the present invention, specifically determining the absence or presence of modified nucleic acid molecules can comprise, consist essentially of, or consist of a nucleic acid amplification step, which helps to maximise the sensitivity of the methods of the present invention. Such amplification techniques are well known in the art and include methods such as PCR, NASBA (Compton, 1991), 3SR (Fahy et al., 1991), rolling circle replication, transcription-mediated amplification (TMA), strand displacement amplification (SDA) (Clinical Chemistry 45:777-784, 1999), the DNA oligomer self-assembly process described in U.S. Pat. No. 6,261,846 (incorporated herein by reference), ligase chain reaction (LCR) (Barringer et al., 1990), selective amplification of target polynucleotide sequences (U.S. Pat. No. 6,410,276), arbitrarily primed PCR (WO 90 / 06995), consensus sequence primed PCR (U.S. Pat. No. 4,437,975), invertor technology, strand displacement technology, and nick displacement amplification (WO 2004 / 067726). The above list is not intended to be exhaustive: any nucleic acid amplification technique can be used provided that the appropriate nucleic acid product is specifically amplified.

[0079] Similarly, sequencing-based methodologies may, in some embodiments, be employed to include any of a range of next-generation sequencing platforms, such as sequencing-by-synthesis of clonally amplified sequences (Illumina), pyrosequencing, 454 sequencing (Roche), nanopore sequencing (e.g., Oxford Nanopore), Ion Torrent (ThermoFisher), and single molecule real-time (SMRT) sequencing (Pacific Biosystems). The fact that novel nucleic acid molecules are generated means that sequencing approaches can confirm the presence or otherwise of modified nucleic acid molecules, as well as provide quantification of the molecules.

[0080] Amplification is achieved by using amplification primers specific to the sequence of the modified nucleic acid molecule to be detected. Primer binding sites corresponding to appropriate regions of the sequence can be selected to provide specificity for the nucleic acid molecule. Those skilled in the art will understand that nucleic acid molecules may also contain sequences other than primer binding sites necessary for detecting novel nucleic acid molecules generated by modification activity in a sample, such as RNA polymerase binding sites or promoter sequences, which may be required for isothermal amplification techniques such as NASBA, 3SR, and TMA.

[0081] One or more primer binding sites can bridge the ligation / extension boundary of the substrate nucleic acid molecule, such that an amplification product is generated only if, for example, ligation / extension occurs. Alternatively, primers can bind to either side of the ligation / extension boundary and amplify directly across the boundary, such that an amplification product is generated (exponentially) only when a ligated / extended nucleic acid molecule is formed. The primers and substrate nucleic acid molecule(s) can be designed to avoid non-specific amplification (e.g., amplification of genomic DNA in a sample).

[0082] The primers may incorporate synthetic nucleotide analogs as appropriate, or may be RNA or PNA based, for example, or mixtures thereof. Primers may be labeled with fluorescent labels and / or FRET pairs, etc., depending on the detection mode employed.

[0083] Probes may be utilized, again, optionally labeled. Detection methods may require the use of nucleotide probes in addition to or as a substitute for primers. For example, branched DNA assays that do not require the use of primers may be employed in some embodiments.

[0084] In certain aspects, the methods of the present invention are carried out using nucleic acid amplification techniques to detect modified nucleic acid molecules produced as a direct result of the action of a nucleic acid modifying activity on a substrate nucleic acid molecule, which is indicative of the presence of a microorganism in a sample. In certain embodiments, the technique used is selected from PCR, NASBA, 3SR, TMA, SDA, and DNA oligomer self-assembly.

[0085] Detection of the amplification products can be performed by routine methods such as, for example, gel electrophoresis, but in some embodiments is performed using real-time or end-point detection methods.

[0086] Several techniques for real-time or end-point detection of the products of amplification reactions are known in the art. These include the use of intercalating fluorescent dyes such as SYBR Green I (Sambrook and Russell, Molecular Cloning—A Laboratory Manual, 3rd Edition), allowing the yield of amplified DNA to be estimated based on the amount of fluorescence produced. Many real-time detection methods generate a fluorescent readout that can be continuously monitored; specific examples include molecular beacons and fluorescence resonance energy transfer probes. Real-time and end-point techniques are advantageous because they maintain the reaction in a "single-tube" environment. This means that downstream analysis is not required to obtain results, allowing for more rapid results. Furthermore, maintaining the reaction in a "single-tube" environment reduces the risk of cross-contamination and allows for quantitative production from the methods of the present invention. This may be particularly important in the context of the present invention, where health and safety concerns may be paramount (e.g., in detecting potential microbial infections in patient samples).

[0087] Real-time and end-point quantification of PCR reactions can be achieved using the TaqMan® system (Applied Biosystems). See Holland et al., "Detection of specific polymerase chain reaction product by utilizing the 5'-3' exonuclease activity of Thermus aquaticus DNA polymerase," Proc. Natl. Acad. Sci. USA 88:7276-7280 (1991); Gelmini et al., "Quantitative polymerase chain reaction-based homogeneous assay with flurogenic probes to measure C-Erb-2 oncogene amplification," Clin. Chem. 43:752-758 (1997); and Livak et al., "Towards fully automated genome-wide polymorphism screening," Nat. Genet. 9:341-342 (1995) (incorporated herein by reference). This type of probe is sometimes commonly referred to as a hydrolysis probe. Suitable hydrolysis / Taqman probes for use in real-time or end-point detection are also provided. The probes may be suitably labelled, for example using the labels detailed below.

[0088] For Molecular Beacon systems, see Tyagi & Kramer, "Molecular beacons - probes that fluoresce upon hybridization," Nat. Biotechnol. 14, 303-308 (1996), and Tyagi et al., "Multicolor molecular beacons for allele discrimination," Nat. Biotechnol. 16, 49-53 (1998), incorporated herein by reference. Beacons are hairpin-shaped probes with an internally quenched fluorophore whose fluorescence is restored upon binding to a target. These probes are sometimes called hairpin probes.

[0089] An additional real-time fluorescence-based system that can be incorporated into the methods of the invention is the Scorpion system; see Detection of PCR products using self-probing amplicons and fluorescence by Whitcombe et al., Nature Biotechnology 17:804-807 (August 1, 1999). Additional real-time or end-point detection technologies well known to those of skill in the art and commercially available include Lightcycler® technology, Amplifuour® primer technology, DzyNA primers (Todd et al., Clinical Chemistry 46:5, 625-630 (2000)), or Plexor™ qPCR and qRT-PCR systems.

[0090] Thus, in a further aspect of the invention, the products of nucleic acid amplification are detected using real-time or end-point techniques. In a specific embodiment of the invention, the real-time technique comprises using any one of hydrolysis probes (Taqman® system), FRET probes (Lightcycler® system), hairpin primers (Amplifluor® system), hairpin probes (Molecular Beacons system), hairpin probes incorporated into primers (Scorpion® probe system), primers incorporating a DNAzyme complementary sequence and a cleavable fluorescent DNAzyme substrate (DzYNA), Plexor qPCR, and oligonucleotide blocking systems.

[0091] The amplification products can be quantified to provide an approximation of the nucleic acid modifying activity of the microorganism in the sample, and therefore the level of the microorganism in the sample. Thus, "absence or presence" is intended to encompass quantification of the level of the microorganism in the sample.

[0092] The inventors have further discovered that the optimal temperature for measuring the nucleic acid modifying activity of a microorganism may not be the same as the optimal temperature for lysing the microorganism. Thus, in some embodiments, lysis of the microorganism is performed at a temperature lower than the step of incubating the lysate with a nucleic acid molecule that acts as a substrate for the nucleic acid modifying activity of the microorganism. As previously discussed, in some embodiments of the present invention, the substrate nucleic acid molecule is included in the lysis reagent used to lyse the microorganism. Such embodiments are consistent with different temperature preferences. Thus, even if the substrate nucleic acid molecule may be included in the lysis reagent, the initial lower temperature does not adversely affect subsequent incubation at a higher temperature where the substrate is modified by the nucleic acid modifying activity released from the microorganism. Thus, in some embodiments, the method involves a step of lysing the microorganism in which the lysis reagent contains a nucleic acid molecule that acts as a substrate for the nucleic acid modifying activity of the microorganism. This step is performed at a temperature lower than the subsequent step of incubating the lysate with the substrate nucleic acid molecule to allow the activity of the enzyme released from the microorganism. Thus, the substrate is exposed to an initial lower temperature, followed by exposure to a higher temperature where the enzyme activity is increased.

[0093] In some embodiments, the step of incubating the lysate with a nucleic acid molecule that acts as a substrate for a nucleic acid modifying activity of the microorganism is carried out at a temperature of at least about 30° C. The temperature can optionally be between about 30° C. and 40° C. or between about 32° C. and 37° C., e.g., about 37° C.

[0094] In further or alternative embodiments, the step of lysing the microorganism is performed at a temperature of about 30° C. or less, optionally about 15° C. to 30° C. or about 18° C. to 25° C., e.g., about 18, 19, 20, 21, 22, 23, 24, or 25° C. In some embodiments, all steps prior to incubating the lysate with a nucleic acid molecule that acts as a substrate for a nucleic acid modifying activity of the microorganism are performed at a temperature of about 30° C. or less. The temperature can optionally be about 15° C. to 30° C. or about 18° C. to 25° C., e.g., about 18, 19, 20, 21, 22, 23, 24, or 25° C.

[0095] Such methods may incorporate any one or more to all of the embodiments described with respect to the various aspects of the present invention.

[0096] In some embodiments, the method is further characterized in that the step of incubating the lysate with the substrate nucleic acid molecule is carried out at a temperature of at least about 30°C, optionally between about 30°C and 40°C, or between about 32°C and 37°C, e.g., about 37°C.

[0097] In further or alternative embodiments, each step prior to incubating the lysate with the substrate nucleic acid molecule is performed at a temperature of about 30°C or less, optionally between about 15°C and 30°C, or between about 18°C ​​and 25°C, e.g., about 18, 19, 20, 21, 22, 23, 24, or 25°C.

[0098] As introduced above, prior to the step of incubating the sample with magnetic particles to form particle-microorganism complexes (i.e., prior to step (a)), the method may include a step of selectively lysing non-microbial cells in the sample while retaining intact microorganisms present in the sample.

[0099] Selectively lysing non-microbial cells in a sample while preserving intact microorganisms present in the sample can include adding a combination of a detergent and one or more enzymes to the sample. The one or more enzymes can include a proteinase and / or a DNAase, and optionally, the proteinase is proteinase K.

[0100] Selective lysis of non-microbial cells in a sample while preserving any intact microorganisms present in the sample prevents enzymatic activity from non-microbial cells, such as white blood cells, which may falsely indicate the presence of microorganisms in the sample. Such selective lysis can be achieved by any suitable means, as discussed further herein. Any suitable reagent can be used that lyses non-microbial cells, particularly mammalian cells, present in the sample but does not lyse the microorganisms in the sample. In some embodiments, the reagent may include a surfactant or detergent, such as a non-ionic surfactant. Suitable examples include polyethylene glycol sorbitan monolaurate (Tween 20), e.g., 1-5% w / v, preferably about 1%. The reagent may include, e.g., 1-5% w / v, preferably about 1% saponin. The reagent may include, e.g., 8.5 g / L of a metal halide salt, such as sodium chloride. The reagent may include a mixture of all three components. The sample can be mixed with the reagent under appropriate conditions to ensure lysis of non-microbial cells, particularly mammalian cells, if present in the sample, but little or no lysis of microbial cells, if present in the sample. The sample can be exposed to the reagent for approximately 5-30 minutes, such as 5, 10, 15, 20, 25, or 30 minutes. This step can be performed at any suitable temperature, e.g., 15-30°C or room temperature. Selective lysis of non-microbial cells can lyse substantially all non-microbial cells present. Selective lysis of non-microbial cells can also lyse some, but not all, of the microbial cells present in the sample, particularly if the microorganisms are fragile and / or have been exposed to high concentrations of antibiotics.

[0101] In some embodiments, according to all aspects of the present invention, selective lysis of non-microbial cells in a sample while preserving any microorganisms present in the sample intact involves adding a combination of a detergent and one or more enzymes to the sample. Without wishing to be bound by theory, detergents selectively permeate the cell membranes of non-microbial cells, while microorganisms are protected by their cell walls. Enzymes are useful for degrading released intracellular material and other cellular debris and may help prevent carryover of released enzyme activity. In some embodiments, the one or more enzymes include a proteinase and / or a nuclease. Suitable proteinases include proteinase K. Suitable nucleases include DNAse. In one embodiment, the reagent used to selectively lyse non-microbial cells includes a combination of Triton X-100 and proteinase K. More specifically, the lysis reagent may include 0.25% Triton X-100 and 4.8 μg / mL proteinase K.

[0102] When lysing non-microbial cells, it is important to inactivate any associated enzyme activity that is released. The present inventors have devised a method that utilizes high pH conditions to ensure effective inactivation of enzyme activity. Microbial cells typically remain intact during at least part of the treatment, and intracellular enzyme activity is not significantly adversely affected by high pH treatment. In addition, the present inventors have previously shown that microbial enzymes are more resistant to high pH treatment in all cases.

[0103] Thus, after selectively lysing non-microbial cells in the sample while preserving any microorganisms present in the sample intact, the method can include exposing the lysate to high pH conditions. The exposure period to high pH conditions is typically less than 20 minutes, and can be 10, 9, 8, 7, 6, or 5 minutes or less, and can be about 5, 6, 7, 8, 9, or 10 minutes. In some embodiments, the treatment is carried out for about 2 to 15 minutes, e.g., about 5 minutes. "About," in this context, means plus or minus 30 seconds.

[0104] Any suitable reagent can be used to provide the high pH conditions. In certain embodiments, the high pH conditions include contacting the sample with an alkali or buffer. In certain embodiments, NaOH or Na2CO3 is used. In specific embodiments, the concentration of NaOH or Na2CO3 is about 5 mM or greater. The buffer can have a pKa value greater than 9. Examples of suitable buffers include borate, carbonate, and pyrophosphate buffers.

[0105] High pH conditions typically inhibit the activity of nucleic acid-modifying enzymes, including ATP-dependent ligases and polymerases, from non-microbial sources, such as mammalian cells, but do not inhibit the activity of microbial ligases or polymerases. This is primarily due to the differential lysis conditions employed in the method, which ensure that only non-microbial enzymes are exposed to high pH conditions. However, it may also be due to the greater tolerance of microbial enzymes to these conditions. A "high pH" is generally a pH of at least about 10, such as about 10, 11, 12, 13, or 14. A "low pH" is generally a pH less than or equal to about 4, such as about 4, 3, 2, or 1. "About," in this context, means 0.5 pH units on either side of the stated value. As will be readily understood by those skilled in the art, altering the pH of a sample can be achieved using any suitable means. Microbial enzymes, such as polymerases and ligases, may be tolerant of extreme pH, while the corresponding mammalian enzymes may be inactivated under the same pH conditions. This facilitates the selective detection of microbial enzyme activity in samples containing both mammalian and microbial cells. In specific embodiments, conditions that inhibit the activity of non-microbial nucleic acid modifying activities, such as ATP-dependent ligases, from mammalian cells, but not microbial sources of nucleic acid modifying activities, such as microbial ligases, include treating the sample with sodium hydroxide (NaOH) or sodium carbonate (NaCO). Such agents, as demonstrated herein, increase the pH of the sample to a high pH while leaving microbial (fungal and bacterial) enzymes active, and thus can be readily used to inactivate non-microbial enzyme activity. Appropriate concentrations and volumes of appropriate agents can be applied by those skilled in the art. However, in certain embodiments, the NaOH is at least about 5 mM NaOH. In some embodiments, the alkali concentration is 10 mM or less, such as 5, 6, 7, 8, 9, or 10 mM.

[0106] In further embodiments, the pH is about 12, which inactivates mammalian nucleic acid-modifying activity (e.g., polymerase and / or ATP-dependent ligase activity) but does not inactivate microbial nucleic acid-modifying activity (e.g., polymerase and / or ligase activity). In specific embodiments, the pH conditions can be increased to at least about 11, or at least 11.2. This treatment can result in lysis of the microorganisms in the sample after a certain period of time, thus releasing the nucleic acid-modifying activity (e.g., polymerase and / or ligase) into the sample. Thus, in some embodiments, lysis of the microorganisms is achieved by high pH treatment. This allows for the detection of nucleic acid-modifying activity (e.g., polymerase and / or ligase) in the sample derived from the microorganisms without the need for a separate cell lysis step. Under such conditions, mammalian ligases (such as blood ATP-dependent ligases) are inactivated. However, typically, the method includes a separate step for lysing the microorganisms in the sample, as discussed in more detail herein.

[0107] In some embodiments, treatment under high pH conditions is stopped by adding a reagent to lower the pH. This is done before the microorganisms are lysed. Suitable reagents include buffers and / or acids. Thus, the pH can be lowered by adding a neutralizing buffer. In a specific embodiment, the buffer comprises a Tris-HCl buffer (e.g., pH 7.2 or 8). Other suitable agents for lowering the pH include acids such as hydrochloric acid (HCl) and sulfuric acid (H2SO4). These (and other) acids can be incorporated into buffers, as will be readily understood by those skilled in the art. One specific reagent useful for treating a sample after the pH has been increased comprises a combination of ammonium sulfate, magnesium sulfate heptahydrate, potassium chloride, and Tris-HCl. More specifically, the reagent can comprise 10 mM ammonium sulfate, 2 mM magnesium sulfate heptahydrate, 10 mM potassium chloride, and 20 mM Tris-HCl [pH 8.0].

[0108] The methods of the present invention can be used to complement any already available diagnostic techniques, potentially as a way to confirm an initial diagnosis. Alternatively, these methods can be used as preliminary diagnostic methods themselves, providing a rapid and convenient diagnostic tool. Furthermore, due to their inherent sensitivity, the methods of the present invention require minimal sample volume, thus preventing unnecessary invasive surgery. Large, but unconcentrated, samples can also be effectively tested according to the methods of the present invention.

[0109] The method of the present invention may involve identifying the nature of the infection once a positive presence of a microorganism has been detected in a sample. Any suitable method may be employed for this further identification step. Compositions and Kits In the course of carrying out the method of the present invention, new compositions (combinations of ingredients) are made. All aspects and embodiments described with respect to other aspects of the present invention (particularly the method) apply mutatis mutandis to the relevant compositions.

[0110] Thus, the present invention provides a composition comprising: a) a sample containing an antimicrobial agent and a viable microorganism; and b) coated particles capable of forming complexes with the viable microorganism in the sample.

[0111] Similarly, the present invention provides a composition comprising a sample containing viable microorganisms complexed with an antimicrobial agent and coated particles.

[0112] In the composition, the sample is as defined herein. Thus, a sample containing viable microbial cells can further contain non-microbial cells. The non-microbial cells can include blood cells. The blood cells can include red blood cells and / or white blood cells.

[0113] The present invention further provides kits for carrying out any of the methods described herein. All aspects and embodiments described with respect to other aspects of the invention (in particular the methods) apply mutatis mutandis to the relevant kits.

[0114] The present invention provides a kit comprising: a) a container containing coated particles capable of forming complexes with viable microorganisms; b) a container containing a medium suitable for incubating and / or culturing viable microorganisms recovered with the coated particles, the container not containing an antimicrobial agent and / or a substance capable of binding to the antimicrobial agent; and / or c) a container containing a wash buffer for washing the coated particles recovered from a sample, the wash buffer not lysing the viable microorganisms.

[0115] In the kit, components a), b), and c) can be in separate containers. The containers included in the kits of the present invention can be any suitable containers for the associated reagents, as will be understood by those skilled in the art. Typically, the containers contain reagents in amounts or concentrations sufficient for performing the method of the present invention once. Thus, the kit can be a single-use kit. The container containing the coated particles capable of forming complexes with viable microorganisms is a container of a material and volume suitable for containing the coated particles. In some embodiments, the container is also of a material and volume suitable for incubating a sample with the coated particles to form particle-microorganism complexes. This means that a sample can simply be added to the container containing the coated particles to begin the method. The container containing the culture medium is a container of a material and volume suitable for containing the culture medium. In some embodiments, the container is also of a material and volume suitable for further containing recovered viable microorganisms, particularly in the form of particle-microorganism complexes. Thus, the culture medium containing container can be suitable for incubating or culturing particle-microorganism complexes. Alternatively, the culture medium can be added to another container (typically of a larger volume) already containing particle-microorganism complexes. The container containing the wash buffer is a container of a material and volume suitable for containing the wash buffer. Typically, the wash buffer is used to wash the particle-microorganism complexes contained in a separate container. Thus, the wash buffer may be provided in a container designed solely for containing the wash buffer. If multiple washes (e.g., two or three) are required, the kit may contain multiple individual containers containing an amount of wash buffer suitable for one wash. Alternatively, a single container may be provided containing sufficient wash buffer for multiple washes (e.g., two or three).

[0116] Suitable washing buffers are described herein. In the kit, the washing buffer may be detergent-free. In some embodiments, the washing buffer contains Tris and / or sodium chloride (suitable concentrations are described in the Examples herein). Typically, the washing buffer is used to remove non-microbial cells in the washing step, and therefore, the washing buffer does not lyse non-microbial cells.

[0117] Any additional means included in the kit may, as appropriate, be contained in one or more containers.

[0118] The kit may further comprise means for detecting and / or characterizing the recovered viable microorganisms. Any suitable means may be employed, which may represent a complete set of reagents necessary for detecting and / or characterizing the viable microorganisms.

[0119] In certain embodiments, the detection means comprises or is a nucleic acid molecule that acts as a substrate for a nucleic acid modifying activity of a microorganism. Suitable substrate molecules in particle DNA oligonucleotide substrate molecules are described herein, and this discussion applies mutatis mutandis.

[0120] In some embodiments, the detection means includes or further includes reagents for nucleic acid amplification. The reagents for nucleic acid amplification may include a primer pair and / or at least one probe. In some embodiments, the primers and / or probes hybridize with microbial nucleic acid molecules. Thus, they can detect microorganisms in a sample by detecting the amplified microbial nucleic acid molecules. Alternatively, the primers or probes hybridize to nucleic acid molecules that act as substrates for the nucleic acid modifying activity of the microorganisms. Such nucleic acid molecules are described in further detail herein.

[0121] The kit may include: (a) coated particles capable of (selectively) forming complexes with microorganisms (i.e., particle-microorganism complexes); and (b) a detection means for detecting the absence or presence of the microorganism in the particle-microorganism complex. The detection means may include a nucleic acid molecule that acts as a substrate for the nucleic acid-modifying activity of the microorganism. The nucleic acid molecule may be at least partially double-stranded and may optionally contain uracil residues in the complementary strand. The complementary strand may contain a base (e.g., dideoxycytidine) at its 3' end that blocks DNA polymerase-mediated second-strand extension. The nucleic acid molecule may be any of the nucleic acid molecules described herein.

[0122] Thus, the kit may further comprise: a) a nucleic acid molecule that acts as a substrate for the nucleic acid modifying activity of the viable microorganism; or b) primers and / or probes that specifically hybridize to (extracted) nucleic acid from the viable microorganism.

[0123] The invention also provides kits comprising: a) a container containing coated particles capable of forming complexes with viable microorganisms, and b) a nucleic acid molecule that acts as a substrate for a nucleic acid-modifying activity of the viable microorganism; or a primer and / or probe that specifically hybridizes to nucleic acid from the viable microorganism. The following discussion applies mutatis mutandis to the related methods.

[0124] According to all aspects of the present invention, the nucleic acid from a live microorganism can be a DNA molecule, e.g., a gene, or an RNA molecule, e.g., mRNA or miRNA. Preferred targets are genes such as ILV3 discussed herein. Other preferred targets are virulence genes, such as those encoding Shiga toxin (e.g., in enterohemorrhagic E. coli infections).

[0125] According to all aspects of the present invention, nucleic acid molecules that act as substrates for nucleic acid modifying activities are described elsewhere herein. Suitable substrates are discussed in more detail herein. By "substrate" is meant a nucleic acid molecule that is (directly) acted upon by an enzyme derived from a microorganism. Typically, such nucleic acid molecules are oligonucleotide substrates. They are synthetic nucleic acid molecules.

[0126] According to all aspects of the present invention, primers and / or probes that specifically hybridize to nucleic acids from viable microorganisms can enable determination of whether bacteria or fungi are present in a sample and / or whether the bacteria or fungi carry antimicrobial resistance genes. Kits can include multiple primer pairs and / or multiple probes for the detection and / or characterization of microorganisms. Multiple primer pairs and / or multiple probes can enable the detection and / or characterization of bacteria and fungi. Primers and / or probes can enable identification of the genus and / or species of microorganisms present in a sample.

[0127] According to all aspects of the present invention, by "specifically hybridize" or equivalent language, it is meant that the primer hybridizes to the target nucleic acid but does not hybridize (or cross-react) with other nucleic acids. The primers and / or probes may hybridize to specific subregions within a gene. The primers and / or probes may specifically hybridize to the same gene of multiple species within the same genus, allowing for identification of the genus of the microorganism. Alternatively, the primers and / or probes may specifically hybridize to a gene of a single species of microorganism, allowing for identification of the species of the microorganism.

[0128] In accordance with all aspects of the present invention, as described in WO 2018 / 189502 (incorporated herein by reference), the ILV3 gene is particularly useful for detecting whether fungi or yeast are present in a sample due to its lack of sequence identity with the human genome.

[0129] According to all aspects of the invention, for detecting or characterising fungi / yeasts, primers may comprise: a. Forward and reverse primers that specifically hybridize to the ILV3 gene of the following Candida species: i. Candida albicans ii. Candida dubliniensis iii. Candida tropicalis iv. Candida parapsilosis v. Candida glabrata vi. Candida krusei vii. Candida guilliermondii viii. Candida auris and optionally comprising, consisting essentially of, or consisting of the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2, respectively; b. Forward and reverse primers that specifically hybridize to the ILV3 gene of Aspergillus species i. Aspergillus fumigatus ii. Aspergillus niger iii. Aspergillus flavus and optionally comprising, consisting essentially of, or consisting of the nucleotide sequences of SEQ ID NOs: 70 and 71, or SEQ ID NOs: 73 and 74, respectively; c. forward and reverse primers that specifically hybridize to the ILV3 gene of Candida albicans, and optionally comprising, consisting essentially of, or consisting of the nucleotide sequences of SEQ ID NOs: 4 and 5, or SEQ ID NOs: 6 and 7, respectively; d. forward and reverse primers that specifically hybridize to the ILV3 gene of Candida dubliniensis, and optionally comprising, consisting essentially of, or consisting of the nucleotide sequences of SEQ ID NOs: 8 and 9, SEQ ID NOs: 10 and 11, or SEQ ID NOs: 12 and 13, respectively; e. forward and reverse primers that specifically hybridize to the ILV3 gene of Candida tropicalis, and optionally comprising, consisting essentially of, or consisting of the nucleotide sequences of SEQ ID NOs: 14 and 15, or SEQ ID NOs: 16 and 17, respectively; f. forward and reverse primers that specifically hybridize to the ILV3 gene of Candida parapsilosis, and optionally comprising, consisting essentially of, or consisting of the nucleotide sequences of SEQ ID NOs: 18 and 19, or SEQ ID NOs: 20 and 21, respectively; g. forward and reverse primers that specifically hybridize to the ILV3 gene of Candida glabrata, and optionally comprising, consisting essentially of, or consisting of the nucleotide sequences of SEQ ID NOs: 22 and 23, SEQ ID NOs: 24 and 25, SEQ ID NOs: 26 and 27, or SEQ ID NOs: 28 and 29, respectively; h. forward and reverse primers that specifically hybridize to the ILV3 gene of Candida krusei, and optionally comprising, consisting essentially of, or consisting of the nucleotide sequences of SEQ ID NOs: 30 and 31, SEQ ID NOs: 32 and 33, SEQ ID NOs: 34 and 35, SEQ ID NOs: 36 and 37, or SEQ ID NOs: 38 and 39, respectively; i. forward and reverse primers that specifically hybridize to the ILV3 gene of Candida guilliermondii, and optionally comprising, consisting essentially of, or consisting of the nucleotide sequences of SEQ ID NOs: 40 and 41, SEQ ID NOs: 42 and 43, SEQ ID NOs: 44 and 45, or SEQ ID NOs: 46 and 47, respectively; j. forward and reverse primers that specifically hybridize to the ILV3 gene of Candida auris, and optionally comprising, consisting essentially of, or consisting of the nucleotide sequences of SEQ ID NOs: 48 and 49, SEQ ID NOs: 50 and 51, SEQ ID NOs: 52 and 53, SEQ ID NOs: 54 and 55, SEQ ID NOs: 56 and 57, SEQ ID NOs: 58 and 59, SEQ ID NOs: 60 and 61, SEQ ID NOs: 62 and 63, SEQ ID NOs: 64 and 65, SEQ ID NOs: 66 and 67, or SEQ ID NOs: 68 and 69, respectively; k. forward and reverse primers that specifically hybridize to the ILV3 gene of Aspergillus fumigatus, and optionally comprising, consisting essentially of, or consisting of the nucleotide sequences of SEQ ID NOs: 76 and 77, SEQ ID NOs: 79 and 80, SEQ ID NOs: 82 and 80, or SEQ ID NOs: 83 and 84, respectively; l. forward and reverse primers that specifically hybridize to the ILV3 gene of Aspergillus niger, and optionally comprising, consisting essentially of, or consisting of the nucleotide sequences of SEQ ID NOs: 87 and 86, respectively; m. Forward and reverse primers that specifically hybridize to the ILV3 gene of Aspergillus flavus, and optionally comprising, consisting essentially of, or consisting of the nucleotide sequences of SEQ ID NOs: 90 and 89, or SEQ ID NOs: 90 and 92, respectively; and / or n. Forward and reverse primers that specifically hybridize to the ILV3 gene of Cryptococcus neoformans, and optionally comprising, consisting essentially of, or consisting of the nucleotide sequences of SEQ ID NOs: 93 and 94, SEQ ID NOs: 96 and 97, SEQ ID NOs: 99 and 100, SEQ ID NOs: 102 and 103, or SEQ ID NOs: 105 and 106, respectively.

[0130] According to all aspects of the invention, for detecting or characterising fungi / yeasts, probes may comprise: a. A probe that specifically hybridizes to the ILV3 gene of the following Candida species: i. Candida albicans ii. Candida dubliniensis iii. Candida tropicalis iv. Candida parapsilosis v. Candida glabrata vi. Candida krusei vii. Candida guilliermondii viii. Candida auris and optionally comprising, consisting essentially of, or consisting of the nucleotide sequence of SEQ ID NO: 3; b. a probe specific for the ILV3 gene of Candida albicans, and optionally comprising, consisting essentially of, or consisting of the nucleotide sequences of SEQ ID NOs: 116 and 117; c. a probe that specifically hybridizes to the ILV3 gene of Candida dubliniensis, and optionally comprising, consisting essentially of, or consisting of the nucleotide sequences of SEQ ID NOs: 118, 119, and 120; d. a probe that specifically hybridizes to the ILV3 gene of Candida tropicalis, and optionally comprising, consisting essentially of, or consisting of the nucleotide sequence of SEQ ID NO: 121; e. a probe that specifically hybridizes to the ILV3 gene of Candida parapsilosis, and optionally comprising, consisting essentially of, or consisting of the nucleotide sequence of SEQ ID NOs: 122 and 123; f. a probe that specifically hybridizes to the ILV3 gene of Candida glabrata, and optionally comprising, consisting essentially of, or consisting of the nucleotide sequence of SEQ ID NO: 124, 125, 126, or 127; g. A probe that specifically hybridizes to the ILV3 gene of Candida krusei, and optionally comprising, consisting essentially of, or consisting of the nucleotide sequence of SEQ ID NO: 128, 129, 130, 131, or 132; h. a probe that specifically hybridizes to the ILV3 gene of Candida guilliermondii, and optionally comprising, consisting essentially of, or consisting of the nucleotide sequence of SEQ ID NO: 133, 134, or 135; i. forward and reverse primers that specifically hybridize to the ILV3 gene of Candida auris, and optionally comprising, consisting essentially of, or consisting of the nucleotide sequence of SEQ ID NO: 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, or 146, respectively; j. A probe that specifically hybridizes to the ILV3 gene of Aspergillus species i. Aspergillus fumigatus ii. Aspergillus niger iii. Aspergillus flavus and optionally comprising, consisting essentially of, or consisting of the nucleotide sequence of SEQ ID NO: 72 or 75; k. A probe that specifically hybridizes to the ILV3 gene of Aspergillus fumigatus, and optionally comprising, consisting essentially of, or consisting of the nucleotide sequences of SEQ ID NOs: 78, 81, and 85; l. A probe that specifically hybridizes to the ILV3 gene of Aspergillus niger, and optionally comprising, consisting essentially of, or consisting of the nucleotide sequence of SEQ ID NO: 88; m. a probe that specifically hybridizes to the ILV3 gene of Aspergillus flavus, and optionally comprising, consisting essentially of, or consisting of the nucleotide sequence of SEQ ID NO: 91; and / or n. A probe that specifically hybridizes to the ILV3 gene of Cryptococcus neoformans, and optionally comprising, consisting essentially of, or consisting of the nucleotide sequence of SEQ ID NO: 95, 98, 101, 104, or 107.

[0131] According to all aspects of the present invention, for detecting and / or characterizing bacteria, primers and probes can specifically hybridize to a specific portion of the 16S region of bacterial DNA. The primers can amplify a specific portion of the 16S region of bacterial DNA. Primers PLK1 (5-TACGGGAGGCAGCAGT-3, SEQ ID NO: 108) and PLK2 (5-TATTACCGCGGCTGCT-3, SEQ ID NO: 109) are highly conserved in different groups of eubacteria. These primers synthesize a 187-bp fragment. PLK2 can be internally labeled with fluorescein. The probe can distinguish between Gram-negative and Gram-positive bacteria. The fluorescent dye-labeled hybridization probes ISN2 (5-CCGCAGAATAAGCACCGGCTAACTCCGT-3, SEQ ID NO: 110) and ISP2 (5-CCTAACCAGAAAGCCACGGCTAACTACGTG-3, SEQ ID NO: 111) emit at different wavelengths (640 and 705 nm) and can be used for detection of bacterial DNA and Gram stain differentiation by fluorescent signal. Other suitable primers may contain the nucleotide sequences CAACGCGAAGAACCTTACC (SEQ ID NO: 112) and ACGTCATCCCCACCTTCC (SEQ ID NO: 113). A suitable Gram-positive probe contains the nucleotide sequence 5'-FAM-ACGACAACCATGCACCACCTG-TAMRA-3' (SEQ ID NO: 114). A suitable Gram-negative probe contains the nucleotide sequence 5'-HEX-ACGACAGCCATGCAGCACCT-TAMRA-3' (SEQ ID NO: 115). The probes are differentially labeled to allow for differential detection, although one of skill in the art will appreciate that other approaches described herein can be employed to facilitate detection.

[0132] Thus, the kit may include a) a container containing coated particles capable of forming a complex with a viable microorganism, and b) a nucleic acid molecule that acts as a substrate for the nucleic acid modifying activity of the viable microorganism; or primers and / or probes that specifically hybridize to the yeast ILV3 gene and / or the bacterial 16S rRNA gene.

[0133] The kit may further comprise a reagent that selectively lyses non-microbial cells in the sample while preserving intact microorganisms present in the sample.

[0134] The kit may further comprise: i) sodium polyanethole sulfonate; and ii) at least one reagent that selectively lyses non-microbial cells in a sample while preserving intact microorganisms present in the sample. Suitable reagents are known in the art and are further discussed herein. As shown herein, sodium polyanethole sulfonate is useful for recovering microorganisms from samples containing non-microbial cells.

[0135] The kit can further include i) sodium polyanethole sulfonate; and ii) a surfactant. A surfactant is an example of a reagent that selectively lyses non-microbial cells in a sample while preserving intact microorganisms present in the sample.

[0136] In view of the increased susceptibility of microorganisms to lysis upon exposure to antimicrobial agents, in some embodiments, the kit may not include a reagent that selectively lyses non-microbial cells. Because the coated particles preferentially bind microorganisms, there is no absolute requirement to lyse non-microbial cells in the sample. However, it may be advantageous to perform one or more washing steps to remove non-microbial cells (typically intact non-microbial cells) and / or cell lysates from the particle-microorganism complexes. Any suitable washing solution may be employed, and examples are discussed herein.

[0137] The kit may include: a) particles capable of forming a complex with a microorganism; b) sodium polyanethole sulfonate; c) at least one reagent that selectively lyses non-microbial cells in a sample while retaining intact microorganisms present in the sample; and d) a detection means for detecting the absence or presence of the microorganism in the particle-microorganism complex, wherein the detection means comprises a nucleic acid molecule that acts as a substrate for a nucleic acid-modifying activity of the microorganism, the nucleic acid molecule being at least partially double-stranded and containing uracil residues in the complementary strand.

[0138] The (substrate) nucleic acid molecule can be designed based on the nucleic acid modifying enzyme comprising a DNA or RNA polymerase, preferably a DNA-dependent DNA polymerase. In some embodiments, the DNA polymerase is DNA polymerase I. In additional or alternative embodiments, the nucleic acid modifying enzyme comprises a ligase, e.g., an ATP- or NAD-dependent ligase.

[0139] The detection means may further comprise reagents for nucleic acid amplification, optionally the reagents for nucleic acid amplification comprise a primer pair and / or at least one probe that hybridises to the nucleic acid molecule.

[0140] The kit may further include a reagent capable of lysing the microorganism in the particle-microorganism complex. Optionally, the reagent capable of lysing the microorganism in the particle-microorganism complex includes a nucleic acid molecule that acts as a substrate for the nucleic acid-modifying activity of the microorganism. Suitable examples include polyethylene glycol sorbitan monolaurate (Tween 20), for example, at 1-5% w / v, preferably about 1%. The reagent may include, for example, 1-5% w / v, preferably about 1% saponin.

[0141] The kit may further include a reagent that selectively lyses non-microbial cells in the sample while preserving intact microorganisms present in the sample.

[0142] The reagent that selectively lyses non-microbial cells in a sample while preserving intact microorganisms present in the sample can include one or more enzymes, which optionally include a proteinase and / or a DNAase. Suitable detergents and enzymes are discussed herein.

[0143] The kit may further include a high pH reagent, such as a base or buffer. This may be, for example, NaOH, e.g., 5 mM NaOH. Other suitable reagents are described herein.

[0144] The kit can further include a neutralization buffer, which can restore the pH of the sample after high pH treatment. Suitable reagents are described herein.

[0145] The nucleic acid modifying enzyme may include (a) a DNA or RNA polymerase, optionally where the DNA polymerase is a DNA-dependent DNA polymerase such as DNA polymerase I; and / or (b) a ligase, optionally where the ligase is an ATP and / or NAD-dependent ligase.

[0146] Additional Features Applicable to All Aspects As already mentioned, the present invention is particularly applicable to clinical samples, in particular samples taken from subjects suspected of suffering from a microbial infection. Thus, according to all aspects of the present invention, the microorganism that may be recovered (and / or characterized) from the sample may be a pathogenic microorganism, such as a pathogenic bacterium or fungus / yeast. The bacterium or fungus / yeast may be any bacterium or fungus / yeast that is capable of causing infection or disease in a subject, preferably a human subject. In one embodiment, the bacteria comprise, consist essentially of, or consist of Staphylococcus spp., such as Staphylococcus epidermidis and Staphylococcus aureus (and preferably methicillin-resistant strains), Enterococcus spp., Streptococcus spp., such as Streptococcus pneumoniae, Pseudomonas spp., Klebsiella spp., particularly Mycobacterium tuberculosis, Vibrio spp., particularly Vibrio cholerae, Salmonella and / or Escherichia coli. The bacteria can comprise, consist essentially of, or consist of Clostridium species, particularly, in certain embodiments, Clostridium difficile (C. difficile). Clostridium difficile is a leading cause of antibiotic-associated diarrhea and colitis, a healthcare-associated enteric infection that primarily affects elderly patients with other underlying conditions. The bacteria can comprise, consist essentially of, or consist of Pseudomonas species, particularly Pseudomonas aeruginosa. The bacteria can comprise, consist essentially of, or consist of Klebsiella species, particularly Klebsiella pneumoniae. In one embodiment, the fungus / yeast can comprise or consist essentially of any one or more of Candida species, Aspergillus species, Cryptococcus species, Histoplasma species, Pneumocystis species, and / or Stachybotrys species.

[0147] As described herein, microorganisms can be detected through their enzyme activity.Therefore, this method provides an indication of the viable microorganisms recovered from the sample containing antibiotics.After a certain period, if microorganisms cannot survive, enzyme activity will be lost from the sample.This represents the advantage of using enzyme activity as an indicator of microorganisms in sample, over the use of nucleic acid molecules, particularly DNA, which can last much longer in some embodiments.

[0148] The methods of the present invention can involve identifying the nature of the infection once a positive presence of a microorganism is detected in a sample. As discussed herein, any suitable method can be used for this further identification step.

[0149] "Antimicrobial agent" is defined herein to encompass any agent that kills or inhibits the growth of microorganisms. The antimicrobial agent may be an antibiotic or an antifungal agent. The antimicrobial agent may be any antimicrobial agent routinely used in the treatment of bloodstream infections. The antimicrobial agent used to treat a subject is selected by a care provider based on the subject's clinical evaluation. This information can be used in practicing the present invention when considering which microorganisms are most likely to be present in the sample. This information can contribute to the selection of appropriate conditions, such as appropriate selective lysis conditions. For example, if the sample is likely to contain more hardy microorganisms, a detergent can be used for selective lysis. On the other hand, if the sample is likely to contain more sensitive microorganisms, a detergent cannot be employed and a wash step can be used instead. The antimicrobial agent can be broad-spectrum, capable of killing or inhibiting a wide range of microorganisms. Non-limiting examples of antibacterial agents include penicillin, meropenem, flucloxacillin, ampicillin, oxacillin, piperacillin-tazobactam, vancomycin, teicoplanin, daptomycin, tigecycline, quinupristin / dalfopristin, gentamicin, amikacin, linezolid, azithromycin, clarithromycin, ciprofloxacin, levofloxacin, sparfloxacin, gatifloxacin, garenoxacin, gemifloxacin, moxifloxacin, doxycycline, TMP-SMX, polymyxin B, cefotaxime, cefotetan, cefamandole, cefuroxime, ceftizoxime, ceftazidime, cefixime, cefoperazone, cefepime, cefazolin, cefoxitin, and ceftriaxone. Non-limiting examples of antifungal agents include flucytosine, fluconazole, itraconazole, voriconazole, posaconazole, etoconazole, griseofulvin, amphotericin B, caspofungin, micafungin, and anidulafungin. The dosage of these agents is determined according to standard care procedures for the particular agent, which contributes to the level of antibacterial agent present in the sample.The antimicrobial agent may be present in the sample at a concentration of at least 0.05, 0.5, 5, 10, 25, 50, 75, or 100 μg / mL. The antimicrobial agent may be present in the sample at a concentration of up to or less than 100, 250, 500, or 1000 μg / mL. The antimicrobial agent may be present in the sample at a concentration of up to or less than 100 μg / mL. The antimicrobial agent may typically be present in the sample at a concentration of 0.5 to 250 μg / mL. The antimicrobial agent may be present in the sample at a concentration of 0.5 to 100 μg / mL.

[0150] As described herein, the present invention is applied in clinical situations where a subject suspected of having a microbial infection is treated with an antimicrobial agent before a body fluid (especially blood) sample is collected. Therefore, the antimicrobial agent given to the subject as treatment may (and should) be the only source of antimicrobial agent in the sample. Therefore, the antimicrobial agent is typically selected by a care provider (e.g., a doctor or nurse) and administered based on the current clinical symptoms demonstrated by the subject. Therefore, the microorganisms in the sample may (or are believed to be) susceptible to the antimicrobial agent.

[0151] A "sample" in the context of the present invention is one that contains or is suspected of containing microorganisms, such as fungi (e.g., yeast) and / or bacteria, and also contains an antimicrobial agent. Typically, the sample is a liquid sample. Thus, the sample can include, consist essentially of, or consist of a clinical sample, such as a bodily fluid sample. A preferred sample type is blood, including whole blood, plasma, serum, and blood-containing samples, including, for example, blood cultures or blood broth. In some embodiments, the sample includes blood, cerebrospinal fluid (CSF), synovial fluid, urine, or bronchoalveolar lavage (BAL). The sample can be a clinical sample taken from a subject undergoing or having undergone treatment with an antimicrobial agent. The sample can include a sample from a patient suspected of having or being screened for an infection. The sample can be any suitable volume, such as 0.2 to 10 ml, or 1 to 10 ml.

[0152] The sample used depends on various factors, such as availability, convenience, and the condition being tested. Typical samples that can be used, but are not intended to limit the invention, include whole blood, serum, plasma, platelets, synovial fluid, and urine samples taken from a patient, most preferably a human patient. The patient may be suspected of having or being screened for a bloodstream infection. The patient may be hospitalized. Samples can be taken from subjects containing more than 5, 10, or 15 million white blood cells (WBCs) per ml of blood.

[0153] In addition to the antimicrobial agent, the sample may further comprise one or more inhibitors of subsequent analysis, which may be selected from blood cell remnants, hemoglobin, leukocyte DNA, and platelets.

[0154] Thus, the present invention also separates a microorganism from one or more inhibitors of microbial growth.

[0155] For the avoidance of doubt, the methods of the present invention represent in vitro methods. They are performed on samples removed from a subject. However, in less preferred embodiments, the methods may additionally comprise (as a preliminary step) the step of obtaining a sample from a subject. Methods for obtaining a suitable sample from a subject are well known in the art. Typically, however, the methods can be performed starting from a sample already isolated from the patient in a separate procedure. The methods are most preferably performed on samples from humans, although the methods of the present invention may have utility for a number of animals. Antimicrobial agents are frequently used in veterinary practice and agriculture.

[0156] The sample may contain non-microbial cells at a concentration of 20,000 to 5 million cells per milliliter. The sample may contain non-microbial cells at a concentration of at least about 100,000 cells per milliliter. Preferably, the sample may contain non-microbial cells at a concentration of at least about 20,000 cells per milliliter.

[0157] The particles used in the present invention are coated. The coating is typically a polymer coating. Alternatively, the coated particles may lack a polymer coating. The coated particles may be coated with molecules such as citrate, starch, mannose-binding lectin, or poly-L-lysine. The molecules coating the coated particles may be adsorbed to the surface of the particles or may form an amalgam with the particles. The coated particles may be fully or partially coated.

[0158] Thus, coated particles are distinguished from uncoated particles. Uncoated particles include particles composed of metals or metal compounds (e.g., metal oxides) that do not have other molecules (e.g., citrate) adsorbed to their surfaces or amalgamated with metals or metal compounds. Coated particles can bind to a variety of microorganisms and form particle-microorganism complexes. Thus, they are "pan-microbial" or "universal" in microbial specificity. Coated particles are preferably magnetic particles and can bind to microorganisms through nonspecific binding (as opposed to the use of target-specific ligands such as antibodies). Coated particles typically have a higher affinity for microorganisms than non-microbial cells.

[0159] The coated particles act by removing microorganisms from the sample, but the antimicrobial agent does not form a significant part of the complex formed, and therefore the coated particles do not substantially bind to the antimicrobial agent.

[0160] Coated particles typically have a polymeric outer surface. The polymeric surface can be a regular outer polymeric surface (within normal manufacturing tolerances) that coats the particle surface (relatively) uniformly. The polymeric surface can include a carbon-based polymer. The polymeric surface can include polyimide, poly(ethylene glycol), polyvinyl alcohol, polyethyleneimine, and polyvinylamine, polyacrylate, polyacrylamide, polyamide, polyester, polycarbonate, polyvinyl, polystyrene, and derivatives and copolymers of any combination thereof. Preferably, the polymeric surface includes polystyrene and / or poly(styrene / divinylbenzene) or polyacrylamide. Most preferably, the polymeric surface includes polystyrene and / or poly(styrene / divinylbenzene).

[0161] The polymer surface may include polydimethylsiloxane, polyimide, polyethylene terephthalate, polymethyl methacrylate, polyurethane, polyvinyl chloride, polystyrene polysulfone, polycarbonate, polymethylpentene, polypropylene, polyvinylidine fluoride, polysilicon, polytetrafluoroethylene, polysulfone, acrylonitrile butadiene styrene, polyacrylonitrile, polybutadiene, poly(butylene terephthalate), poly(ether sulfone), poly(ether ether ketone), poly(ethylene glycol), styrene-acrylonitrile resin, poly(trimethylene terephthalate), polyvinyl butyral, polyvinylidene difluoride, poly(vinylpyrrolidone), and derivatives and copolymers of any combination thereof. In some embodiments, the polymer surface is not silicone-based.

[0162] In certain embodiments according to all aspects of the present invention, the coated particles may include any one or more of i) carboxylic acid groups; ii) amino groups; iii) hydrophobic groups; and iv) streptavidin on the outer surface. These functional groups have been shown by the inventors to be useful in universal viable microbial recovery. Such functional groups typically form part of the polymer coating and are not provided by a separate targeting ligand. Thus, in some embodiments, i) carboxylic acid groups, ii) amino groups, or ii) hydrophobic groups may not be part of a polypeptide or peptidomimetic. Similarly, i) carboxylic acid groups, ii) amino groups, or ii) hydrophobic groups may not be part of a nucleoside, nucleotide, or nucleic acid. Similarly, i) carboxylic acid groups, ii) amino groups, or ii) hydrophobic groups may not be part of a lipid, steroid, hormone, or cofactor.

[0163] The coated particles may be, and in fact typically are, magnetic. Magnetic beads are paramagnetic and are attracted to an externally applied magnetic field. Magnetic beads are well known and commercially available. The coated particles may be superparamagnetic. The coated particles may comprise a metal or metal compound. The coated particles may comprise iron oxide. The iron oxide may comprise magnetite and / or maghemite. The iron oxide may comprise Fe 2+ and Fe 3+ The iron oxide may not comprise a 1:1, 2:1, 3:1, or 4:1 ratio of iron oxide. Preferably, the iron oxide is encapsulated by a polymer coating. The coated particles may be an amalgam of iron oxide and a polymer. The coated particles may be partially encapsulated by an outer polymer surface, although this is less preferred. The coated particles may comprise a core and a polymer coating. The core may be magnetic. The coated particles may comprise iron oxide coated with citrate, starch, mannose-binding lectin, or poly-L-lysine.

[0164] The coated particles may have a diameter of 0.05 to 20 μm, or 0.05 to 1 μm, for example, 0.1 to 0.5 μm, or 0.2 to 0.3 μm. Preferably, the coated particles have a diameter of 0.2 to 0.3 μm. The coated particles may have a diameter of 0.1 to 3 μm or 0.1 to 2 μm. More preferably, the coated particles have a diameter of 0.1 to 1.0 μm.

[0165] The coated particles are (a) a polymer coating (e.g., polystyrene, poly(styrene / divinylbenzene), or polyacrylamide); (b) a polymer coating (e.g., polystyrene, poly(styrene / divinylbenzene) or polyacrylamide) having any one of the following additional groups on its surface: carboxylic acid, amine, or streptavidin; or c) surface-attached citrate, mannose-binding lectin, starch, or poly-L-lysine molecules may include:

[0166] the coated particles are magnetic; a) polymer coatings (such as polystyrene, poly(styrene / divinylbenzene) or polyacrylamide); b) a polymer coating (e.g., polystyrene, poly(styrene / divinylbenzene) or polyacrylamide) having any one of the following additional groups on its surface: carboxylic acid, amine or streptavidin; or c) surface-attached citrate, mannose-binding lectin, starch, or poly-L-lysine molecules may include:

[0167] The coated particles may be metals (such as iron) or metal compounds (such as iron oxide), and a) polymer coatings (such as polystyrene, poly(styrene / divinylbenzene) or polyacrylamide); b) a polymer coating (e.g., polystyrene, poly(styrene / divinylbenzene) or polyacrylamide) having any one of the following additional groups on its surface: carboxylic acid, amine or streptavidin; or c) surface-attached citrate, mannose-binding lectin, starch, or poly-L-lysine molecules may include:

[0168] Thus, the coated particle has an outer polymer surface. In some embodiments, the outer surface of the coated particle may not be coated with any of (i) antibodies, (ii) carbohydrates, (iii) peptides derived from apolipoprotein H protein, (iv) mannose-binding lectin protein, (v) polyamines, or (vi) cationic surfactants. The mannose-binding lectin (MBL) protein may be a genetically engineered protein based on MBL. For example, it may be a genetically engineered protein containing the pathogen-binding portion of MBL fused to the Fc region of an immunoglobulin (i.e., FcMBL).

[0169] In some embodiments, the exterior surface of the coated particle may not be coated with (i) an antibody, (ii) a carbohydrate, or (iii) an innate immune system protein.

[0170] In some embodiments, the outer surface of the coated particle may not be coated with any of: (i) an antibody, (ii) a carbohydrate, (iii) a peptide derived from apolipoprotein H protein, (iv) a mannose-binding lectin, or (v) an agglutinating agent (e.g., an agglutinating agent defined in WO 03 / 102184).

[0171] In some embodiments, the exterior surface of the coated particle may not be coated with any of: (i) antibodies, (ii) carbohydrates, (iii) innate immune system proteins, or (iv) aggregating agents (e.g., aggregating agents defined in WO 03 / 102184).

[0172] In some embodiments, the exterior surface of the coated particle may not be coated with polylysine or polylysine-like moieties.

[0173] The antibody may be a fragment or derivative of an antibody that retains antigen-specific binding function, including Fab fragments, ScFv, single domain antibodies, nanobodies, heavy chain antibodies, etc.

[0174] The carbohydrate can be a monosaccharide, an oligosaccharide (eg, a disaccharide or trisaccharide), a polysaccharide, and / or a derivative thereof.

[0175] The outer surface of the coated particle may not be coated with a ligand. The outer surface of the coated particle may not be coated with a non-specific ligand (e.g., the non-specific ligands described in WO 01 / 53525). The outer surface of the coated particle may not be coated with a non-proteinaceous ligand (e.g., the non-proteinaceous ligands described in WO 01 / 53525).

[0176] The exterior surface of the coated particles may be carboxylated.

[0177] The exterior surface of the coated particle may be coated with streptavidin. The exterior surface of the coated particle may be coated with streptavidin and not coated with a ligand.

[0178] In accordance with all relevant aspects and embodiments of the present invention, the term "sodium polyanetholesulfonate" is intended to encompass all functionally equivalent derivatives and salt forms thereof (e.g., potassium polyanetholesulfonate, magnesium polyanetholesulfonate, sodium amylosulfate, etc.).

[0179] As used herein, "coating" or "coated" generally refers to a layer of molecules or material formed on the outermost or exposed layer of a particle. While the coating may be partial, the coating need not include a continuous layer of molecules on the outermost or exposed layer of the particle.

[0180] Throughout this disclosure, the terms "particles" and "beads" may be used interchangeably. [Brief explanation of the drawings]

[0181] [Figure 1] Images of agar plates at T=0 hours (left) and T=2 hours (right), both showing a central zone of no growth due to inhibition by antibiotics from the sample (see Example 1). [Figure 2] 1 is an image of blood samples showing the extent of blood lysis for each sample set: E-BUF, UREA, Tris+NaCl, frozen (left to right) (see Example 9). [Figure 3] This is an image of the final sample production before PCR setup. This image visually demonstrates the benefits of SPS for sample processing with magnetic beads in blood. SPS appears to allow for more thorough removal of blood components, as indicated by less red eluate in the presence of SPS. Note that the BacTec PLUS aerobic broth used in the blood broth sample set also contains SPS (see Example 10). [Example]

[0182] The invention will be understood with reference to the following non-limiting examples.

[0183] [Experimental section] Abbreviations and definitions: 5th% 5th percentile threshold calculation to determine 5% FPR (Formula = PERCENTILE.INC(array,0.05)) ABX antibiotic or antibacterial agent BO Bros Only BB Blood Broth CBA Columbia Blood Agar Cfu colony forming units COL Columbia basal agar Confirm: A PCR multiplex assay targeting microbial DNA according to Gram status (Gram-negative, Gram-positive, or Candida). CPD Citrate Phosphate Dextrose Ct cycle threshold CV Critical Value (cfu): Formula: Sample cfu ÷ 2 ΔCt Theoretical detection limit based on cfu value and ΔCt using D1.. Dilution point (10x series) Dil Dilution DMBB Detergent-Free Microbial Binding Buffer DWB Detergent-free washing buffer E-Buff Blood Lysis Buffer E * cfu Extrapolated cfu value using the dilution point with the highest countable TVC in the dilution series EC Escherichia coli ETGA enzyme template generation and amplification FA+ Resin, FA Plus BioMerieux GMBB Aerobic Blood Culture Bottles with Mild Microbial Binding Buffer IPC Internal Process Control: PCR template present in the LM to demonstrate correct sample processing and verify PCR amplification for ETGA-negative samples LAWN Confluent microbial growth LM Microbial Lysis Mixture containing a mixture of surfactants and microbial lytic enzymes MM Master Mix NoCt: No amplification above threshold fluorescence after 50 cycles No NSC spike control O / n overnight PC polymerase spike control PCR polymerase chain reaction Positivity threshold calculated from Pt NSC / NC results qPCR quantitative polymerase chain reaction RT Room temperature (+19~+20℃) SA Standard Aerobic Blood Culture Bottle, SA BioMerieux SAB Sabouraud dextrose agar s / n supernatant SPS Sodium Polyanethole Sulfonate TNTC Too many to count TVC total number of viable bacteria WB Wash Buffer (containing Tris-HCl + sodium chloride + Igepal + sodium deoxycholate + tergitol unless otherwise specified); or whole blood where indicated ΔCt: The difference between two Ct values ​​(typically NSC Ct - positive sample Ct)

[0184] Example 1 the purpose To evaluate whether microorganisms in clinical blood containing antibiotics can be "rescued" and removed before being inoculated into fresh medium for incubation, and to simply investigate whether the rescued organisms grow more efficiently compared to organisms maintained in antibiotic-containing blood.

[0185] Specifically, we aim to show that when Staphylococcus aureus is inoculated into blood containing an inhibitory concentration of the lytic antibiotic piperacillin and incubated for 2 hours, the organism can be captured on magnetic capture beads and grown by transferring them to blood culture bottles containing no antibiotic.

[0186] material Buffer composition [Table 1]

[0187] method Test organism: Staphylococcus aureus Antibiotic: Piperacillin 96 μg / mL blood Human blood: Cambridge Bioscience, Research Donors, London UK Overnight cultures of the test organisms were prepared in blood / broth medium (50:50 blood:broth medium from BioMerieux SA culture bottles). Serial 10-fold dilutions of overnight cultures were prepared in blood / broth as needed. Antibiotics (abx) were added at the required levels to whole human blood (in citrate-phosphate-dextrose, CPD, anticoagulant). Whole blood was used as an antibiotic-free control. Blood + antibiotics or blood was spiked with 10 μL of organism dilution(s) per mL of blood (enough prepared for experimental requirements). A 100 μL plate count sample was taken (TO sample). The inoculated samples were incubated at 37°C (static) for 2 hours. A 100 μL plate count sample was taken (T2 sample).

[0188] As a control, 5 mL of spiked blood + abx or blood alone was added to a blood culture bottle (BioMerieux SA culture bottle) using a vial adapter (West Pharmaceutical Services, Inc. PA, US) and incubated in an automated blood culture cabinet (adapter removed).

[0189] In the "rescue" test, 5 mL of spiked blood + abx or blood alone was treated with Momentum capture beads (streptavidin coated, approximately 300 nm beads, Bio estapor, Merck catalog number BE-M 08 / 0.3) as follows.

[0190] Momentum capture beads diluted in detergent-free microbial binding buffer (DMBB-Tris+NaCl) - 50 μL beads:700 μL DMBB per cleanup Add 750 µL of diluted beads to a 50 mL conical centrifuge tube Add 5 mL of broth to the tube. Add 5 mL of spiked blood + abx or blood only to the tube. ITL TherMix (Integrated Technologies Ltd. Kent, UK), incubated at 32.5°C / 30 min / 100 rpm - ramp rate protocol Magnetize with a V&P magnet (V&P Scientific Inc., CA, US) for 5 minutes, then remove the supernatant. Resuspend the beads in 1 mL of detergent-free wash buffer (DWB - Tris + NaCl) and transfer to a 2 mL Eppendorf flip-top tube on a DynaMag-2 magnet (Thermo Fisher Scientific, Life Technologies Ltd. Paisley, UK). Magnetize for 5 minutes, then remove the supernatant. Resuspend the beads in 1 mL of DWB from the magnet and place them on the DynaMag-2 magnet. Magnetize for 5 minutes, then remove the supernatant. Resuspend the beads in 1 mL of DWB and add to a fresh, unused blood culture bottle (BioMerieux SA culture bottle) containing standard blood culture medium using a vial adapter and begin incubation (adapter removed). Blood culture bottles are incubated in an automated blood culture cabinet (BacT / ALERT BioMérieux) and the time to positivity is recorded. The "turnover" time is the time for a bottle to become positive as determined by the automated blood culture cabinet. The "inversion" time for each bottle is recorded Record plate count (total viable count TVC)

[0191] result As can be seen in Table 1, the organisms recovered from samples 4b and 5b reverted to positive at 10-15 hours of culture while the control blood culture remained negative at 120 hours (5 days). The rescued organisms continued to grow only until the organisms in the control blood culture turned positive, when the presence of antibiotics killed or inhibited them. Growth occurred in both samples containing no antibiotics, one treated as a control and one treated with the rescue treatment. [Table 2]

[0192] In conclusion, we have demonstrated that organisms can be "rescued" and removed from antibiotic-containing blood samples, inoculated into fresh medium, and incubated until positive. In the experiments examined here, organisms that were not rescued and remained in antibiotic-containing samples did not grow and remained negative at day 5.

[0193] The following examples demonstrate the ability of various coated particles to form particle-microorganism complexes in different samples and under different conditions, thereby recovering viable microorganisms. Various downstream detection and characterization methods are also illustrated.

[0194] Example 2 In the manual format, two bead types were compared: Merck Bio-Estapor (streptavidin conjugated) 300 nm beads (product-BE-M08 / 03; "Bio-Estapor") and Ademtech Bio-Adembeads Streptavidin+ 200 nm beads (product number 03222; "Bio-Ademtech"), with ApoH Technologies Peps6 beads (reference-MP20006; "ApoH Peps6").

[0195] In Experiment 1A, aliquots of Bio-Estapor beads (25 μL) and ApoH Pep6 beads (10 μL) were compared for binding. The larger volume of Bio-Estapor reflects the lower number of beads per mL in the provided material compared to the ApoH material. Three microorganisms were tested: Escherichia coli (a gram-negative bacterium), Staphylococcus epidermidis (a gram-positive bacterium), and Candida albicans (a yeast). 0.5 mL of organism suspension was exposed to the beads in 0.5 mL of "TTGB" microbial binding buffer provided in the ApoH Peps6 kit ("Peps6 Captobac", reference MP10031-50T).

[0196] After allowing the organisms to bind for 30 minutes, the bead sample was separated from the liquid supernatant by applying a magnetic field to concentrate the beads and removing the supernatant with a pipette. The beads were gently washed with three aliquots of wash buffer (50 mM Tris pH 8, 1% v / v Igepal CA-630, 150 mM NaCl, 0.25% v / v Tergitol 15-S-9), and the retained supernatant and washed beads were analyzed for viable organisms by two methods: colony counts on agar Petri dishes and detection of microbial DNA by the enzymatic template generation and amplification (ETGA) test (Zweitzig et al., 2012. Characterization of a novel DNA polymerase activity assay enabling sensitive, quantitative, and universal detection of viable microbes. Nucleic Acids Research, Vol. 40, No. 14, e109, pp. 1-12; and described in WO 2011 / 130584, WO 2013 / 103744, and WO 2016 / 005768).

[0197] Plate counts in Table 1A show that for Bio-Estapor beads and E. coli, the majority of growth was found on the beads (33 CFU) versus the supernatant (2 CFU), similar to the results from ApoH Peps6. No growth was observed for Staphylococcus epidermidis, as this organism did not appear to grow in the original broth. Candida albicans showed approximately 10% and 90% binding of CFU in the supernatant with both Bio-Estapor and ApoH Peps6. These results indicate that Bio-Estapor beads appear to bind organisms at a rate comparable to that of the commercially available Peps6 biobinding beads under the test conditions. The highly sensitive ETGA assay supports this result, but indicates that S. epidermidis may bind more to Bio-Estapor than to Peps6, as indicated by the lower Cq value. [Table 3]

[0198] Experiment 1B demonstrates binding of E. coli under conditions similar to those of Experiment 1A, except that the wash step was omitted in 1B. Experiment 1B shows that another bead, Bio-Ademtech, also binds organisms, albeit at a lower level (see Table 1B). Here, plate counts indicate that approximately one-third of the viable bacterial population bound to the beads. The more sensitive ETGA DNA polymerase assay indicates that half of the organisms remained on the beads, as the Cq and supernatant relative to the beads were approximately equal. [Table 4]

[0199] Example 3 In experiment 3, we compared ApoH Peps6, Bio-Estapor, and Estapor beads (product MI-030 / 40; "Estapor COOH") with a carboxylated surface. A fluorescent ATP assay (BacTiter-Glo Microbial Cell Viability Assay; Promega Corporation, G8230) was used to measure the number of organisms remaining in the supernatant after 30 minutes of E. coli binding to the beads. While this is an indirect test in that it does not directly detect the presence of organisms on the beads, it is a useful comparative test for our ligand-based beads (ApoH Peps6) and non-liganded beads (Bio-Estapor and Estapor COOH). One mL of 10 4 After binding 30 mL of E. coli CFU / mL from phosphate-buffered saline, aliquots of the supernatant were assayed for ATP as a measure of organism content using the BacTiter-Glo assay. The results in Table 2 show that the reduction in organism levels in the supernatants of Peps6 beads, Bio-Estapor, and Estapor COOH, as measured using this technique, was 33%, 27%, and 24%, respectively. [Table 5]

[0200] Example 4 Example 4 shows the results of testing Escherichia coli (EC), Staphylococcus aureus (SA), and Candida albicans (CA) in a dilution series using the automated magnetic separation method described in Example 2. The assay used Bio-Estapor 300 nm diameter beads as the capture medium with a binding buffer of TTGB containing 0.25% Tergitol. Ten-fold dilutions of each of the three organisms were performed, and serial changes in Ct were recorded to generate dose-response curves. [Table 6]

[0201] The following example demonstrates the universal microbial capture of microorganisms by magnetic beads in Momentum's Magnitor test. The Magnitor test consists of two microbial detection readouts.

[0202] ETGA: detection of microbial polymerases from intact microbial cells Confirm: Detection of microbial DNA by Gram status (Gram-negative, Gram-positive, or Candida)

[0203] Key findings: Magnetic beads capture bacteria and fungi from simple buffers and a variety of complex biological specimen types Microbial capture is performed using a variety of different bead sizes (0.2-1.5 μm diameter beads) and surface coatings (e.g., carboxylated, hydrophobic, aminated, etc.). Certain binding buffer components can improve microbial detection in Magnitor assays, for example, detergent-based lysis of blood.

[0204] Example 5: Microbial detection relies on capture by magnetic beads. the purpose: Microbial binding performance was evaluated for Escherichia coli, Staphylococcus aureus, and Candida albicans in a simple Tris+NaCl buffer (pH was buffered at physiological salt concentrations to prevent microbial osmotic shock, which can only occur in water). A "no-beads control" sample set was also included in this experiment to demonstrate that detection relies on the presence of magnetic beads for microbial capture.

[0205] Preparation of magnetic beads: Estapor beads (Merck, catalog number M1-30 / 40) were washed with 3 x 1 mL of 1 x Tris + NaCl buffer: 40 μL beads were resuspended in a final volume of 400 μL of 1 x Tris + NaCl buffer (1% solids).

[0206] Protocol: Overnight liquid cultures of microorganisms were used as the standard in BacTec PLUS aerobic broth (3 mL broth inoculated from an agar plate). The next day (approximately 16 hours later), 1.88 μL of E. coli and S. aureus liquid cultures were added to 3 mL of broth, and 18.75 μL of Candida albicans liquid culture was added to 3 mL of broth. Growth was continued for 2 hours at 37°C and 500 rpm.

[0207] After 2 hours of growth, the microbial precultures were diluted (DF10) in 1× Tris+NaCl buffer (50 mM Tris-HCl [pH 8.0]+150 mM NaCl) to create four dilution points per microorganism.

[0208] A 100 μL TVC was performed for each organism dilution. Manual simulation of the Magnitor performed using a DynaMag-2 magnet and manual liquid transfer: 1 mL of sample was added to a 2 mL tube containing 15 μL of pre-washed beads - note, all microbial samples were diluted in the same 1× buffer, so 112 μL of 1× Tris+NaCl buffer was not added to the tube with the beads (as per standard protocol).

[0209] Shaking (1000 rpm) at 37°C for 30 minutes Magnetized on DynaMag-2 for 5 minutes. All s / n removed 1 mL washing buffer (WB) was added and the tube was mixed (1000 rpm) for 2 min at RT. Magnetized on Dynmag-2 for 3 minutes All s / n removed 50 μL Lysis Mix (LM) was added to the tubes and the magnet was removed. (5 μL of Polymerase Control (PC) was added to each PC sample tube.) ETGA reaction: 5 minutes at 1000 rpm, then 55 minutes at 800 rpm at 26°C Manual qPCR was set up for ETGA and confirmation (10 μL reaction). result: [Table 7] [Table 8] [Table 9] [Table 10] [Table 11] [Table 12]

[0210] analysis: Magnitor results for the "(+)BEADS" samples showed very strong cell density-specific ETGA and Confirm signals for all three microbial species, demonstrating bead-specific binding of a broad range of microbial groups (GrNeg, GrPos, Candida).

[0211] The Candida results were able to follow the cell density trend better, but it should be noted that the liquid culture was entirely particulate, which may have affected the quality of the serial dilutions. Some evidence of microbial cells persists in the "(-)BEADS" control, but is expected with only a single wash step.

[0212] Example 6: Microbial capture from blood by magnetic beads occurs in simple and complex blood lysis buffers, allowing for microbial detection comparable to centrifugation capture the purpose: To develop a simple and rapid "Rapid Magnitor" test (the protocol does not include a wash step), two different blood lysis buffers in two different dilution formats were compared. Test conditions: 2 x EBB 1 mL of 2 x EBB + 1 mL specimen 10x EBB 112µL of 10x EBB + 1mL specimen 2xB-BUF 1mL of 2xB-BUF + 1mL specimen 10x B-BUF 112 μL of 10x B-BUF + 1 mL of specimen 10x EBB: 500 mM Tris-HCl [pH 8.0] + 2.5% Tergitol 10x B-BUF: 500 mM Tris-HCl [pH 8.0] + 1.5 M sodium chloride + 10% Igepal + 5% sodium deoxycholate + 2.5% tergitol

[0213] Sample setup: A 1E-3 dilution of E. coli o / n liquid culture was spiked into blood broth (6.25 μL o / n per mL: 244 μL o / n + 39 mL blood broth) and grown for 60 min in a shaking incubator at 37 °C and 500 rpm. After 2 h of growth, samples were generated by adding 1 mL of specimen to a 2 mL tube containing buffer (and 15 μL of BioEstapor beads (Merck, catalog number BE-M 08 / 0.3) for the Mag Beads sample set): triplicate E. coli (EC) and spike control (NSC) samples per test condition. 100 μL TVCs were performed for NSCs and E. coli (including dilution of specimens to ensure countable plates)

[0214] Protocol: Samples set up as above proceeded immediately to the spin or Mag bead protocol. Spin Protocol Samples were centrifuged for 3 minutes at 9000 x g (tube hinge facing outwards for pellet traceability). The supernatant was removed 50 μL of LM was added to the sample (approximately 10x pipette mix to resuspend the pellet) The samples were placed in a shaking incubator at 900 rpm for 5 minutes and then at 800 rpm for 55 minutes (26°C). Samples were centrifuged at 17,000 × g for 1 minute, and then qPCR was performed. Mag Beads Protocol The samples were placed in a shaking incubator at 900 rpm for 30 minutes (37°C). The samples were placed on a DynaMag-2 magnetic rack for 5 minutes, and then the supernatant was removed. 50 μL of LM was added to the sample (approximately 10x pipette mix to resuspend the pellet) The samples were placed in a shaking incubator at 900 rpm for 5 minutes, then at 800 rpm for 55 minutes (26°C). The samples were magnetized for 3 minutes before setting up the qPCR. Manual qPCR was performed using only the ETGA master mix (10 μL reaction).

[0215] result: [Table 13] [Table 14]

[0216] analysis: Microorganism binding by magnetic beads occurs as follows: Simple and complex blood lysis buffers (EBB = Tris-HCl + Tergitol; B-BUF = Tris-HCl + sodium chloride + Igepal + sodium deoxycholate + Tergitol) were used. The test signal from blood varied depending on the components of the blood lysis buffer. Diluted (2x buffer: 1 part blood lysis buffer to 1 part specimen) and concentrated (10x buffer: 1 part blood lysis buffer to 9 parts specimen) sample formats Furthermore, the microbial detection signal for microbial capture by magnetic beads is comparable to capture by centrifugation.

[0217] Example 7: Microbial capture from blood by magnetic beads is independent of blood lysis, but downstream microbial detection is improved when microbial binding occurs in lysed blood the purpose: Given the recent finding that multiple bead types / sizes yield similar Magnitor results for serial microbial dilutions and NSCs, we suspected that components within Momentum's binding buffer might mediate / promote this observed universal microbial binding characteristic. To investigate this possibility, we performed a dilution series of E. coli bacteria in standard binding buffer (B-BUF) compared to detergent-free B-BUF consisting of only Tris-HCl [pH 8.0] + NaCl to test whether detergents in general are important for microbial binding. Sample sets were prepared in blood broth, broth only, and 1x binding buffer only, and results for the different specimen types were compared.

[0218] Preparation: Freshly prepared 100 mL 10x Binding Buffer: B-BUF: 500 mM Tris-HCl [pH 8.0] + 1.5 M sodium chloride + 10% Igepal + 5% sodium deoxycholate + 2.5% tergitol Tris + NaCl: 500 mM Tris-HCl [pH 8.0] + 1.5 M sodium chloride Estapor beads (Merck, catalog number M1-30 / 40) were washed 3 x 1 mL in the respective 1x buffer (diluted 10x B-BUF or 10x Tris + NaCl): 40 μL beads were resuspended to a final concentration of 400 μL 1x buffer (1% solids).

[0219] Protocol: An o / n liquid culture of E. coli was set as a standard in BacTec PLUS aerobic broth, and then the next day (approximately 16 hours later), 1.88 μL o / n was added to 3 mL of broth (equivalent to an EC 1E-1 dilution added to broth at 6.25 μL / mL) and grown for 2 hours at 37°C and 500 rpm.

[0220] After 2 hours of growth, the E. coli preculture was serially diluted (DF10) to an EC of 1E-6 in either prewarmed blood-broth (BB), broth only (BO), or 1× buffer (B-BUF or Tris+NaCl).

[0221] 100 μL TVC was performed for all E. coli dilutions and NSCs Manual simulation of the Magnitor performed using a DynaMag-2 magnet and manual liquid transfer: 1 mL of sample was added to a 2 mL tube containing 112 μL of binding buffer (either B-BUF or Tris+NaCl: 10× for BB and BO sample sets; and 1× for buffer sample sets) + 15 μL beads (pre-washed in the respective buffer). Shaking (1000 rpm) at 37°C for 30 minutes Magnetized on DynaMag-2 for 5 minutes. All s / n removed 1 mL of WB was added and the tube was mixed (1000 rpm) for 2 min at RT. Magnetized on Dynmag-2 for 5 minutes All s / n removed Add 50 µL of LM to the tube and remove the magnet. (Add 5 µL of polymerase control (PC) to each PC sample tube.) The ETGA reaction was carried out at 26°C for 5 minutes at 1000 rpm, then 55 minutes at 800 rpm. Manual qPCR was set up for ETGA and confirmation (10 μL reaction).

[0222] observation: As expected, no hemolysis was observed in Tris+NaCl. In the absence of surfactant, the beads are more granular / aggregated

[0223] result: [Table 15] [Table 16] [Table 17] [Table 18] [Table 19] [Table 20]

[0224] analysis: Although surfactant is important for obtaining good ETGA results in the presence of blood (as shown by the poor ETGA results for "10x Tris + NaCl with BB"), microbial capture / detection is still evident in the absence of blood lysis (as shown by the results for the "10x Tris + NaCl with BB" sample set).

[0225] The successful ETGA results in the absence of blood indicate that detergent as a component of the binding buffer is not required for E. coli binding to the beads. The successful ETGA results in the "10x Tris + NaCl with 1x Tris + NaCl" sample set demonstrate that biological components in the blood and / or broth are not necessary for microbial binding.

[0226] Interestingly, B-BUF appears to have a slightly inhibitory effect on the ETGA signal in the 10x B-BUF with BO and 1x B-BUF sample sets, an observation that is not unexpected, as other recent studies have shown that sodium deoxycholate can have a somewhat inhibitory effect on this assay. Confirm performed best in the "10x Tris + NaCl with 1x Tris + NaCl" sample set. All other similar sample sets produced similar Confirm GrNeg results.

[0227] As might be expected, the IPC signal was somewhat inhibited by the presence of blood.

[0228] These results demonstrate that neither the surfactant nor the biological sample is a mediator of microbial binding for E. coli.

[0229] Example 8: Microbial capture by magnetic beads occurs in the absence of blood, regardless of pH buffering or osmotic stabilization with salt the purpose: To further explore the importance of Momentum's binding buffer in mediating microbial binding, the effects of pH buffering and salt on binding were examined in a clean system (i.e., in the absence of either blood or broth). 10x buffer preparation: 25 mL of each buffer was freshly made: BUF-1 500mM Tris-HCl[pH7.4]+1.5M NaCl BUF-2 500mM Tris-HCl[pH8.0]+1.5M NaCl BUF-3 500mM Tris-HCl[pH8.5]+1.5M NaCl BUF-4 500mM Tris-HCl [pH 8.0] only BUF-5 1.5M NaCl only BUF-6 Water only Estapor beads (Merck, catalog M1-30 / 40) were washed 3 x 1 mL each in 1x buffer (dilution of 10x buffer): 30 μL beads were resuspended in a final volume of 300 μL of 1x buffer (1% solids).

[0230] Protocol: An o / n liquid culture of E. coli was set as a standard in BacTec PLUS aerobic broth (with SPS) and nutrient broth (NB without SPS), and then the next day (approximately 16 hours later), 1.88 μL o / n was added to 3 mL broth (equivalent to an EC 1E-1 dilution added to broth at 6.25 μL / mL) for each broth type (NB in ​​the morning and PLUS broth in the afternoon) and growth incubation was carried out for 2 hours at 37°C, 500 rpm.

[0231] For each experiment (NB and PLUS), a 1E-1 E. coli preculture was diluted (DF10) to 1E-6 of E. coli in each 1x buffer (BUF-1 to BUF-6). Perform 100 µL TVCs using separate sets of EC dilutions made in the relevant broth (NB or PLUS broth) to prevent plate viability discrepancies arising from different 1x buffers.

[0232] Manual simulation of the Magnitor performed using a DynaMag-2 magnet and manual liquid transfer: 1 mL of sample was added to a 2 mL tube containing 112 μL of each 1× buffer + 15 μL beads (pre-washed in the respective buffer). Shaking (1000 rpm) at 37°C for 30 minutes Magnetized on DynaMag-2 for 5 minutes. All s / n removed 1 mL of WB was added and the tube was mixed (1000 rpm) for 2 min at RT. Magnetized on Dynmag-2 for 5 minutes All s / n removed Add 50 μL of LM to the tube and remove the magnet. The ETGA reaction was carried out at 26°C for 5 minutes at 1000 rpm, then 55 minutes at 800 rpm. Manual qPCR was set up for ETGA and confirmation (10 μL reaction).

[0233] result: [Table 21] [Table 22] [Table 23] [Table 24] [Table 25] [Table 26] [Table 27] [Table 28] [Table 29] [Table 30]

[0234] analysis: All buffers containing only water demonstrated similar E. coli capture (as indicated by similar ETGA and Confirm results) - however, low cell densities in water alone sometimes demonstrated osmotic microbial lysis (BUF-6). Both PLUS broth and NB-grown E. coli produced very similar Magnitor results for all buffers tested, indicating that SPS does not play a distinct role in mediating microbial binding of E. coli. These results indicate that buffer components are not essential for binding of E. coli to Estapor (carboxylated) beads.

[0235] Example 9: Microbial capture from blood by magnetic beads can be performed using a variety of different hemolysis methods the purpose: To determine whether microbial capture and detection can occur when using alternative hemolysis methods.

[0236] Preparation: The binding buffer was prepared as follows: E-BUF = 500 mM Tris-HCl [pH 8.0] + 1.5 M sodium chloride + 10% Igepal + 2.5% Tergitol UREA=83mM Tris-HCl[pH8.0]+10M urea Tris + NaCl = 500 mM Tris-HCl [pH 8.0] + 1.5 M sodium chloride BioEstapor beads (Merck, Cat. No. BE-M 08 / 0.3) were resuspended before use.

[0237] Protocol: S. aureus o / n liquid culture was set as a standard in BacTec PLUS aerobic broth, and then the next day (approximately 16 hours later), 3.0 μL o / n was added to 3 mL of blood broth (1E-3 dilution) and grown for 4 hours at 37°C and 500 rpm.

[0238] After 4 h of growth, the S. aureus preculture was serially diluted to 1E-6 (DF10) in prewarmed blood broth.

[0239] A 100 μL TVC was performed for all S. aureus dilutions and NSCs. Manual sample handling using the DynaMag-2 magnet and manual liquid transfer with a pipette: Initial Setup For samples with urea, 0.25 mL of specimen was added to a 2 mL tube containing 0.75 mL of urea + 15 μL of beads. For samples to be frozen, 1 mL of specimen was added to a 2 mL tube and then quickly frozen on dry ice for 5 minutes. The specimen was thawed at 37°C for 5 minutes, then 112 μL of Tris+NaCl+15 μL beads was added. For samples using E-BUF or Tris+NaCl, 1 mL of specimen was added to a 2 mL tube containing 112 μL of binding buffer (either E-BUF or Tris+NaCl) + 15 μL of beads. Processing of all samples Orbital mixing (1000 rpm) was performed for 30 minutes at 37°C. Magnetize on DynaMag-2 for 5 minutes Remove all s / n Add 1 mL of WB and mix the tube at 37°C for 3 minutes (1000 rpm). Magnetize on Dynmag-2 for 5 minutes Remove all s / n Add 50 μL of LM to the tube and remove the magnet. ETGA reaction: 26°C at 1000 rpm for 5 min, then 800 rpm for 55 min Manual qPCR setup for ETGA and confirmation (10 μL reaction) observation: Hemolysis was observed in frozen samples after thawing (see Figure 2). Near-instant hemolysis was observed with urea Some beads appeared to be lost during treatment of the sample with urea. No obvious hemolysis was observed in the Tris+NaCl sample set (as expected).

[0240] result: [Table 31] [Table 32] [Table 33] [Table 34] [Table 35] [Table 36]

[0241] FIG. 2 shows the extent of hemolysis for each sample set: E-BUF, urea, Tris+NaCl, and frozen (from left to right).

[0242] analysis: Microbial capture and detection of S. aureus by magnetic beads is comparable to alternative lysis methods, as determined by Confirm, without hemolysis. However, microbial detection by ETGA is improved to different degrees by alternative hemolysis methods, due to the effect of reduced blood-borne ETGA signal.

[0243] Example 10: SPS is necessary for optimal bead performance, sample processing, and microbial detection in whole blood the purpose: To determine the optimal SPS concentration for the Magnitor Rapid Test using a 1 mL whole blood sample. A secondary objective was to evaluate the effect of SPS on microbial viability in whole blood as determined by TVC.

[0244] Test conditions: 2 x 5 mL of whole blood or BacTec PLUS aerobic blood broth (1:3 ratio) was dispensed into each sample set (E. coli and NSC samples). SPS was then added as follows: Sample set 10% SPS (μL) No BB WB0% None WB0.01% 5 WB0.02% 10 WB0.04% 20 WB0.06% 30 WB0.08% 40 WB0.10% 50 Next, 5 μL of E. coli 1E-2 preculture was added to each 5 mL sample tube to recreate the standard 1E-5 dilution sample.

[0245] Protocol: 1.88 μL of neat overnight in Nutrient Broth (NB) was added to 3 mL of NB; incubated for 2 hours at 37°C, 500 rpm After 2 hours, the E. coli preculture was diluted 10-fold with warmed NB; then, 5 μL was added to each 5 mL sample tube (prepared as indicated in the test conditions). Magnitor testing was initiated immediately and TVC was performed as detailed below.

[0246] Manual simulation of the Magnitor performed using a DynaMag-2 magnet and manual liquid transfer: Each sample tube was preloaded with 112 μL of E-BUF (500 mM Tris-HCl [pH 8.0] + 1.5 M sodium chloride + 10% Igepal + 2.5% Tergitol) + 15 μL beads (BioEstapor, Merck, catalog number BE-M 08 / 0.3), and then 1 mL of specimen was added to the sample tube. Orbital mixing (1000 rpm) was performed for 30 minutes at 37°C. Magnetized on DynaMag-2 for 5 minutes. All s / n removed 1 mL of WB was added and the tube was mixed (1000 rpm) for 3 min at RT. Magnetized on Dynmag-2 for 5 minutes All s / n removed 50 μL of LM was added to the tube and the magnet was removed. ETGA reaction: 26°C at 1000 rpm for 5 min, then 800 rpm for 55 min Manual qPCR was set up for ETGA and confirmation (10 μL reaction).

[0247] result: TVC analysis 100 μL to COL plate at time zero The sample tube (containing approximately 2 mL of sample) was left on the bench (still) at room temperature (20.4°C). TVC was performed at the time points indicated in the table: samples were thoroughly mixed before inoculation. [Table 37] [Table 38] [Table 39] [Table 40]

[0248] analysis: SPS has shown the benefit of providing microbial protection / viability in whole blood based on the TVC assay, but does not generally pose significant problems for E. coli viability in whole blood.

[0249] The incorporation of SPS improved the sample processing efficiency and microbial detection performance of both ETGA and Confirm readouts. 0.06% SPS provided the best results for TVC-based viability, ETGA detection (best results considering E. coli and NSC sample Ct), and PCR inhibition as indicated by IPC Ct values. Confirm GrNeg results were also improved by adding SPS to whole blood, although the exact concentration of SPS was not critical.

[0250] Example 11: Microbial capture from blood by magnetic beads occurs using various commercially available carboxylated bead products of the same size (approximately 300 nm diameter) the purpose: To compare alternative carboxylated magnetic beads of similar size using Momentum's Magnitor assay.

[0251] Test conditions: [Table 41]

[0252] Protocol: E. coli and S. pyogenes overnight liquid cultures were set as standards in 3 mL broth and blood broth (BacTec PLUS aerobic), respectively, and incubated for 16–20 h (37°C).

[0253] the next day: A liquid culture of E. coli was diluted 1E-3 with blood broth and then spiked into blood broth (6.25 μL per mL of blood broth) and pre-incubated for 1 hour and 30 minutes (37°C). A liquid culture of S. pyogenes was diluted 1E-1 with blood broth and then spiked into blood broth (6.25 μL per mL of blood broth) and pre-incubated for 2 hours and 30 minutes (37°C).

[0254] The E. coli experiment conducted in the morning A 1E-3 preculture of E. coli was serially diluted in blood broth to generate five dilution points (1E-3 to 1E-7). Samples were set up by adding 1 mL specimen to a 2 mL tube pre-loaded with 15 μL of 1% solids beads + 112 μL of binding buffer; and Magnitor V4.0 testing was performed: 5 dilution points per bead type with 3 bead types + 3 NSCs (8 sample sets) were tested on each epMotion 5073m.

[0255] Streptococcus pyogenes experiment conducted in the afternoon A 1E-3 preculture of S. pyogenes was serially diluted in blood broth to generate five dilution points (1E-1 to 1E-5). Samples were set up by adding 1 mL specimen to a 2 mL tube pre-loaded with 15 μL of 1% solids beads + 112 μL of binding buffer; and Magnitor V4.0 testing was performed: 5 dilution points per bead type with 3 bead types + 3 NSCs (8 sample sets) were tested on each epMotion 5073m.

[0256] Magnitor V4.0 Protocol (Automated Sample Processing on the epMotion 5073m) Orbital mixing (1000 rpm) was performed for 30 minutes at 37°C. Magnetized for 15 minutes 1 mL of s / n was removed While the beads were magnetized, 0.82 mL of WB was added to the tube. 1 mL of s / n was removed While the beads were magnetized, 50 μL of LM was added to the tube. The magnetization was turned off and the ETGA reaction was carried out: 5 min at 1000 rpm, then 55 min at 800 rpm at 26°C. qPCR was set up for ETGA and confirmation (10 μL reaction).

[0257] result: An internal positivity threshold (Pt) was calculated for each bead type using NSC (n=6): formula = PERCENTILE.INC(array, 0.05) [Table 42] [Table 43] [Table 44]

[0258] observation: Beads B were difficult to resuspend before dilution to 1% solids and appeared more diluted visually after dilution to 1% solids. At the end of the process, the samples were placed on a DynaMag-2 magnetic rack and all bead types CG were magnetized identically, apart from bead A, which appeared to have a heavy pellet, and bead B, which had a very small bead pellet.

[0259] analysis: All carboxylated magnetic beads tested here demonstrate microbial binding as determined by ETGA and Confirm readouts. However, the sensitivity of microbial detection varies somewhat depending on the level of blood-borne ETGA signal and / or assay inhibition.

[0260] Example 12: Microbial capture from blood by magnetic beads occurs using a variety of different bead sizes and functional coatings the purpose: To compare the microbial capture performance of various commercially available magnetic beads with different sizes and functional coatings using Momentum's Magnitor test (ETGA and Confirm technology). Two experiments were performed to demonstrate microbial capture for automated (Protocol 1) and manual (Protocol 2) sample processing. Importantly, Protocol 2, which includes three bead resuspension washes, more convincingly demonstrates that the ETGA / Confirm signal is specific to bead-bound microbial cells rather than sample carryover (in contrast to Protocol 1, which includes a single-bead magnetized wash step).

[0261] Test conditions: [Table 45] Sample setup (run separately for protocols 1 and 2 and performed on different days): An o / n liquid culture of E. coli was set as the standard in 3 mL broth (BacTec PLUS aerobic) and incubated for 16–20 h (37°C). The next day, the liquid culture of E. coli was diluted 1E-3 with blood broth and subjected to a 2-hour growth incubation (37°C at 500 rpm). After 2 hours of growth incubation, a 1E-3 preculture of E. coli was serially diluted in blood broth to generate three dilution points (EC 1E-6 to 1E-8). One mL of specimen (three E. coli dilutions and three NSC samples: six sample sets) was added to a 2 mL sample tube pre-loaded with 112 μL of 10× E-BUF (500 mM Tris-HCl [pH 8.0] + 1.5 M sodium chloride + 10% Igepal + 2.5% Tergitol) + 15 μL beads (1% solids), and then the Magnitor test was initiated according to either Protocol 1 or Protocol 2 (see below).

[0262] Protocol 1 (automated sample processing on the epMotion 5073m): Orbital mixing (1000 rpm) was performed for 30 minutes at 37°C. Magnetized for 15 minutes 1 mL of s / n was removed While the beads were magnetized, 0.82 mL of WB was added to the tube. 1 mL of s / n was removed While the beads were magnetized, 50 μL of LM was added to the tube. The magnetization was turned off and the ETGA reaction was carried out: 5 min at 1000 rpm, then 55 min at 800 rpm at 26°C. qPCR was set up for ETGA and confirmation (10 μL reaction).

[0263] Protocol 2 (Manual Sample Processing using a DynaMag-2 magnet and manual liquid transfer with a pipette): Orbital mixing (1000 rpm) was performed for 30 minutes at 37°C. Magnetized on DynaMag-2 for 5 minutes. All s / n removed 1 mL of WB was added and the tube was mixed (1000 rpm) for 2 min at RT. Magnetized on Dynmag-2 for 5 minutes All s / n removed 1 mL of WB was added and the tube was mixed (1000 rpm) for 2 min at RT. Magnetized on Dynmag-2 for 5 minutes All s / n removed 1 mL of WB was added and the tube was mixed (1000 rpm) for 2 min at RT. Magnetized on Dynmag-2 for 5 minutes All s / n removed 50 μL of LM was added to the tube and the magnet was removed. The ETGA reaction was carried out at 26°C for 5 minutes at 1000 rpm, then 55 minutes at 800 rpm. Manual qPCR was set up for ETGA and confirmation (10 μL reaction).

[0264] result: [Table 46] [Table 47]

[0265] analysis: These results demonstrate that a variety of different bead sizes and functional coatings produce comparable levels of microbial binding as determined by ETGA and Confirm readouts.

[0266] Example 13: Magnetic beads of different sizes and functional coatings can be used to capture a wide range of microbial species (Gram-negative, Gram-positive, and Candida) from blood the purpose: To compare the microbial capture performance of various commercially available magnetic beads with different sizes and functional coatings using Momentum's Magnitor test (ETGA and Confirm technology), E. coli was previously tested (Bead Size and Coating I: Source Experiments: 20190221_WP7_Bead-Comparison_Analysis and 20190228_WP7_Bead-Comparison-3-wash_Analysis). To expand on this previous study, three additional microbial species were tested (Staphylococcus aureus, Streptococcus pneumoniae, and Candida albicans).

[0267] Test conditions: [Table 48]

[0268] Protocol: Sample setup: Overnight liquid cultures of microorganisms were set up in BacTec PLUS aerobic broth (3 mL of broth was inoculated from the agar plate). The next day (approximately 16 hours later), 3 mL of blood broth (1E-1 dilution) was inoculated with 300 µL of Streptococcus pneumoniae and Candida albicans liquid cultures, and 3 mL of blood broth (1E-3 dilution) was inoculated with 3 µL of Staphylococcus aureus liquid culture; growth was carried out for 2 hours at 37 °C and 500 rpm.

[0269] After 2 hours of growth, the microbial precultures were diluted (DF10) in blood broth to produce three dilution points per organism.

[0270] A 100 μL TVC was performed for each organism dilution. Manual Simulation of Magnitor and Manual Liquid Transfer Performed with DynaMag-2 Magnet: One mL of specimen (3 dilutions per microbial species and 3 NSC samples: 12 sample sets) was added to a 2 mL sample tube pre-loaded with 15 μL of beads (1% solids) and 112 μL of E-BUF (500 mM Tris-HCl [pH 8.0] + 1.5 M sodium chloride + 10% Igepal + 2.5% Tergitol). Orbital mixing (1000 rpm) was performed for 30 minutes at 37°C. Magnetized on DynaMag-2 for 5 minutes. All s / n removed Add 1 mL of WB and mix the tube (1000 rpm) for 3 min at RT. Magnetized on Dynmag-2 for 5 minutes All s / n removed 50 μL of LM was added to the tube and the magnet was removed. The ETGA reaction was carried out at 26°C for 5 minutes at 1000 rpm, then 55 minutes at 800 rpm. Manual qPCR was set up for ETGA and confirmation (10 μL reaction).

[0271] result: [Table 49] [Table 50] [Table 51]

[0272] analysis: These results demonstrate that a variety of different bead sizes and functional coatings produce comparable levels of microbial binding as determined by ETGA and Confirm readouts.

[0273] Example 14: Magnetic beads of different sizes and functional coatings can be used to capture a wide range of microbial species (Gram-negative, Gram-positive, and Candida) from a simple Tris+NaCl buffer the purpose: To compare the microbial capture performance of various commercially available magnetic beads with different sizes and functional coatings using Momentum's Magnitor test (ETGA and Confirm technology). This experiment was performed using a simple buffer (50 mM Tris-HCl [pH 8.0] + 150 mM NaCl) as the sample and a wash buffer, i.e., no surfactant was used before the addition of the microbial lysis mixture.

[0274] Test conditions: [Table 52] Protocol:

[0275] Sample setup: Set up overnight liquid cultures (o / n) of microorganisms in BacTec PLUS aerobic broth (inoculate 3 mL of broth from the agar plate). The next day (approximately 16 h), inoculate 3 mL of broth (1E-3 dilution) with 3 µL of E. coli and S. aureus liquid cultures, and inoculate 3 mL of broth (1E-1 dilution) with 300 µL of Candida albicans liquid culture. Grow for 2 h at 37 °C and 500 rpm.

[0276] After 2 hours of growth, the microbial precultures were diluted (DF10) in 1× Tris+NaCl buffer to produce three dilution points per organism.

[0277] A 100 μL TVC was performed for each organism dilution. Manual simulation of the Magnitor performed using a DynaMag-2 magnet and manual liquid transfer: 1 mL of specimen (3 dilutions per microbial species and 3 NSC samples: 12 sample sets) was added to a 2 mL sample tube pre-loaded with 15 μL of beads (1% solids). Orbital mixing (1000 rpm) was performed for 30 minutes at 37°C. Magnetized on DynaMag-2 for 5 minutes. All s / n removed 1 mL of WB (1x Tris + NaCl) was added and the tube was mixed (1000 rpm) for 3 min at RT. Magnetized on Dynmag-2 for 5 minutes All s / n removed 50 μL of LM was added to the tube and the magnet was removed. The ETGA reaction was carried out at 26°C for 5 minutes at 1000 rpm, then 55 minutes at 800 rpm. Manual qPCR was set up for ETGA and confirmation (10 μL reaction).

[0278] result: [Table 53] [Table 54] [Table 55]

[0279] analysis: These results demonstrate that in a clean system (i.e., simple Tris+NaCl buffer instead of a biological specimen), a variety of different bead sizes and functional surfaces (carboxylation and hydrophobicity) result in comparable levels of microbial binding as determined by ETGA and Confirm readouts.

[0280] Interestingly, aminated beads (NH2-1.5) produced extremely poor Magnitor results for specimen types indicating little to no microbial binding. This observation differs from the situation with blood broth specimens, where aminated beads produced comparable levels of microbial capture to the other beads tested.

[0281] Example 15: Magnetic beads of different sizes and functional coatings can be used to capture a wide range of microbial species (Gram-negative, Gram-positive, and Candida) from unlysed blood the purpose: To compare the microbial capture performance of various commercially available magnetic beads with different sizes and functional coatings in the absence of hemolysis, i.e., without surfactant in the binding buffer, using Momentum's Magnitor test (ETGA and Confirm technology).

[0282] Test conditions: [Table 56]

[0283] Protocol: Sample setup: Set up overnight liquid cultures (o / n) of microorganisms in BacTec PLUS aerobic broth (inoculate 3 mL of broth from the agar plate). The next day (approximately 16 h), inoculate 3 mL of broth (1E-3 dilution) with 3 µL of E. coli and S. aureus liquid cultures, and inoculate 3 mL of blood broth (1E-1 dilution) with 300 µL of Candida albicans liquid culture. Grow for 2 h at 37 °C and 500 rpm.

[0284] After 2 hours of growth, the microbial precultures were diluted (DF10) in blood broth to produce three dilution points per organism.

[0285] A 100 μL TVC was performed for each organism dilution. Manual simulation of the Magnitor performed using a DynaMag-2 magnet and manual liquid transfer: One mL of specimen (3 dilutions per microbial species and 3 NSC samples: 12 sample sets) was added to a 2 mL sample tube pre-loaded with 15 μL of beads (1% solids) and 112 μL of binding buffer (Tris-HCl + sodium chloride). Orbital mixing (1000 rpm) was performed for 30 minutes at 37°C. Magnetized on DynaMag-2 for 5 minutes. All s / n removed 1 mL of WB was added and the tube was mixed (1000 rpm) for 3 min at RT. Magnetized on Dynmag-2 for 5 minutes All s / n removed 50 μL of LM was added to the tube and the magnet was removed. ETGA reaction: 26°C at 1000 rpm for 5 min, then 800 rpm for 55 min Manual qPCR was set up for ETGA and confirmation (10 μL reaction). result: [Table 57] [Table 58] [Table 59]

[0286] analysis: These results demonstrate that a variety of different bead sizes and functional coatings result in comparable levels of microbial binding from blood in the absence of hemolysis, as determined by ETGA and Confirm readouts. However, microbial detection by ETGA is substantially reduced by the increase in NSC ETGA signal in the absence of hemolysis when compared to previous experiments on hemolysis during microbial binding.

[0287] Example 16: Microbial capture by magnetic beads occurs in a variety of complex biological specimen types the purpose: To investigate whether microbial capture using magnetic beads is possible in other complex biological fluids in addition to blood.

[0288] Test conditions: [Table 60]

[0289] Protocol: An E. coli o / n liquid culture was set as a standard in BacTec PLUS aerobic broth, and then the next day (approximately 16 h later), 3 μL o / n was added to 3 mL of broth (E. coli 1E-3) for 2 h of growth incubation at 37°C and 500 rpm.

[0290] After 2 hours of growth, the E. coli 1E-3 preculture was diluted to five serial dilution points (E. coli 1E-6 to 1E-9) for each specimen type. 100 μL of TVC was performed on COL agar plates. Manual simulation of the Magnitor performed using a DynaMag-2 magnet and manual liquid transfer: One mL of specimen was added to a 2 mL sample tube pre-loaded with 112 μL of binding buffer (500 mM Tris-HCl [pH 8.0] + 1.5 M sodium chloride + 10% Igepal + 2.5% Tergitol + 0.5% sodium deoxycholate) + 15 μL of beads (BioEstapor beads; Merck #BE-M08 / 03 (1% solids)). Note: The sample tube for the Tris-NaCl sample set was not pre-loaded with 112 μL of binding buffer (to avoid contamination with detergents that may inhibit microbial growth for the regrowth assay). Shaking (1000 rpm) at 37°C for 30 minutes Magnetized on DynaMag-2 for 5 minutes. All s / n removed Add 1 mL of WB and mix (1000 rpm) for 3 min at RT - Note, 1 mL of Tris+NaCl buffer was added instead of wash buffer for the Tris+NaCl sample set to avoid detergent contamination that may inhibit microbial growth for the regrowth assay. Magnetized on Dynmag-2 for 5 minutes All s / n removed Add 50 μL of LM to the tube and remove the magnet - note, for the regrowth assay the beads were resuspended in 100 μL of Tris+NaCl buffer. The ETGA reaction was carried out at 26°C for 5 minutes at 1000 rpm, then 55 minutes at 800 rpm. Manual qPCR was set up for ETGA and confirmation (10 μL reaction).

[0291] result: [Table 61]

[0292] Regrowth assay of paired Tris+NaCl sample sets 1. 100 μl of Tris+NaCl sample was inoculated / seeded - "sample" 2. 100 μl of supernatant was inoculated / seeded after the microbial binding step - "post-binding" 3. 100 μl of supernatant was plated / inoculated after the washing step - "Post-wash" 4. 50 μL of beads were resuspended in 100 μL of Tris+NaCl buffer and plated / inoculated (i.e., 50% of the material was plated and 50% of the material was inoculated into liquid culture)—"beads" Plates and liquid cultures were incubated overnight at 37°C. [Table 62] [Table 63] [Table 64] Note, there was no observable amplification in the Candida channel of Confirm [Table 65]

[0293] analysis: These results demonstrate that magnetic beads can be used to capture microorganisms from a variety of complex biological specimen types, as determined by ETGA and Confirm readouts.

[0294] Regrowth assays demonstrate that E. coli can be bound to magnetic beads and then regrow on agar and liquid culture as determined by observable growth of the "bead" sample set.

[0295] Example 17: Capture and detection of microorganisms from non-blood specimens is possible in the absence of specimen lysis the purpose: To demonstrate microbial capture and detection in non-blood specimens of milk and urine using non-lytic binding buffer and non-lytic wash buffer.

[0296] Preparation: 10x Tris + NaCl binding buffer = 500 mM Tris-HCl [pH 8.0] + 1.5 M sodium chloride 1x Tris+NaCl wash buffer = 1 in 10 dilution of 10x Tris+NaCl binding buffer Fresh (human) urine Semi-skimmed (pasteurized) milk BioEstapor beads (Merck, Cat. No. BE-M 08 / 0.3) were resuspended before use.

[0297] Protocol: Escherichia coli, Staphylococcus aureus, Candida albicans, and Streptococcus pneumoniae o / n liquid cultures were set as standards in BacTec PLUS aerobic broth. The next day (approximately 16 hours later), 3 μL of E. coli and S. aureus o / n were used to inoculate fresh 3 mL broth cultures (1E-3 dilution), and 300 μL of C. albicans and S. pneumoniae were used to inoculate fresh 3 mL broth cultures (1E-1 dilution), and growth was carried out for 2 hours at 37°C and 500 rpm.

[0298] After 2 hours of growth, the microbial precultures were serially diluted (DF10) in prewarmed fresh urine and fresh milk, resulting in five dilution points: Escherichia coli 1E-5 to 1E-9, Staphylococcus aureus 1E-5 to 1E-9, Candida albicans 1E-2 to 1E-6, and Streptococcus pneumoniae 1E-2 to 1E-6.

[0299] For each microbial dilution tested in milk and urine, 100 μL of TVC was performed, and milk and urine NSCs were plated onto three types of agar plates (SAB, COL, and CBA). One mL of specimen (five dilutions of each microbial species and four NSC samples: 24 samples per specimen type) was added to a 2 mL sample tube preloaded with 112 μL of binding buffer plus 15 μL of beads (1% solids), and then the automated Magnitor test was started.

[0300] Automated sample processing on the epMotion 5073m: Orbital mixing (1000 rpm) was performed for 30 minutes at 37°C. Magnetized for 15 minutes 1 mL of s / n was removed While the beads were magnetized, 0.82 mL of WB (1x Tris + NaCl) was added to the tube. 1 mL of s / n was removed While the beads were magnetized, 50 μL of LM was added to the tube. The magnetization was turned off and the ETGA reaction was carried out: 5 min at 1000 rpm, then 55 min at 800 rpm at 26°C. qPCR was set up for ETGA and confirmation (10 μL reaction).

[0301] result: [Table 66] [Table 67] [Table 68]

[0302] analysis: These results demonstrate that microbial capture by magnetic beads is possible in alternative specimens to blood (particularly urine and milk) in the absence of specimen lysis, as determined by ETGA and Confirm readouts.

[0303] However, the presence of commensal microorganisms in these specimen types (especially milk) affects the level of background signal in the ETGA and Confirm readouts.

[0304] Examples 18 to 25 the purpose To evaluate whether microorganisms in clinical blood, which may or may not contain antimicrobial agents, can be "rescued" and removed before being inoculated into fresh medium for incubation. To investigate whether the rescued organisms grow more efficiently compared to organisms remaining in blood or blood containing antibiotics. The intended use of this method is to enhance the recovery and detectability of microorganisms.

[0305] introduction It is widely known that blood cultures taken from patients already receiving antibiotics often fail to grow organisms, even when the patient is clearly infected. In recent years, major blood culture providers have offered bottles containing antibiotic-absorbent resins, but this has only partially solved the problem. In this study, we aim to demonstrate that when organisms are inoculated into blood containing inhibitory concentrations of antibiotics, they can be captured using our magnetic capture beads and transferred to antibiotic-free blood culture bottles, allowing the microorganisms to grow. A range of organisms and antibiotics were tested.

[0306] Main findings: Although a difference of 2-4 hours was observed in the inversion time between horse and human blood, the final results were comparable. Therefore, subsequent experiments can be performed using horse blood, which is of practical importance during this time when human blood is unavailable (due to the COVID-19 pandemic).

[0307] Sample incubation in the presence of magnetic beads did not aid or hinder the growth of the organisms.

[0308] The turnover times between the different capture reagents for E. coli and S. aureus were very similar, indicating that this rescue method allows for recovery of the organism and can be performed with or without hemolytic agents.

[0309] In standard aerobic (SA) bottles containing bacteria in blood containing antimicrobial agents, rescue methods always reverted to positive before blood culture (BC) controls, which were negative on day 5. For Candida albicans, rescue methods reverted as much as 27 hours faster than BC controls, and for Cryptococcus neoformans, reversal times were over 13 hours faster.

[0310] When different magnetic bead types for organism capture were compared (Example 23), all bead rescue samples reversed positivity within 18 hours compared to unrescued blood culture controls that were negative after 5 days. This was true for all five bead types tested and for both E. coli in blood containing 0.075 mg / L ciprofloxacin and S. aureus in blood containing 500 mg / L vancomycin.

[0311] When five different magnetic bead types were further compared for biocapture (Example 24), all bead rescue samples reverted to positive for Staphylococcus aureus within 17 hours compared to the no-rescue blood culture control, which was negative. This was true for Staphylococcus aureus in blood containing 500 mg / L vancomycin for all six bead types tested. For E. coli, bead rescue samples reverted to positive within 30 hours for all six bead types tested in blood containing 0.075 mg / L ciprofloxacin. Blood culture controls (no bead rescue) were negative.

[0312] As reported by Flayhart et al., 2007, at high peak serum concentrations of antibiotic-blood (ceftriaxone), FA+ blood culture bottles are unable to recover S. pneumoniae. The data presented herein demonstrate that using the methods of the present invention, S. pneumoniae can be recovered and successfully grown from blood containing high peak serum concentrations of antibiotics.

[0313] As reported by Chung et al., 2019, at the high peak serum concentrations of antibiotic-blood, FA+ blood culture bottles are unable to recover organisms. The data presented herein demonstrate that using the methods of the present invention, organisms can be recovered and successfully grown from concentrations as low as 180 cfu / mL from blood containing antibiotics at high peak serum concentrations.

[0314] material Buffer and Reagent Compositions [Table 69]

[0315] Biological, antimicrobial and other materials Blood: Horse (defibrillated) from TCS Biosciences Ltd., Buckingham, UK Test Organism: Gram-positive: Staphylococcus aureus ATCC 25923; Streptococcus pneumoniae ATCC 49619 Gram negative: Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC 13883, Pseudomonas aeruginosa ATCC 27853 Fungi / yeast: Candida albicans ATCC 10231, Cryptococcus neoformans NCPF 8281 Antibiotics: Gram-positive: Vancomycin, piperacillin, cefepime, ceftriaxone for Streptococcus pneumoniae Gram-negative: ciprofloxacin, piperacillin, meropenem, cefotaxime, and cefepime Yeast: Amphotericin B

[0316] Example 18: Rescue of Staphylococcus aureus from blood containing piperacillin the purpose: The purpose of this replicate experiment is to evaluate whether equine blood can substitute for human blood during times when human blood is unavailable.

[0317] Protocol: Day 1: Prepare an overnight culture of the test organism in blood / broth medium (50:50 blood:broth) and incubate at 37° C. in a shaking incubator. Day 2: If necessary, prepare a 10-fold dilution series of the overnight culture in blood / broth. Antibiotics are added to whole blood (defibrated horse blood) at the required levels. Whole blood as a control without antibiotics Add 10 μL (enough prepared for experimental requirements) of the test dilution(s) of the organism per mL of blood to either the blood + antibiotic or blood. A 100 μL plate count sample is taken (TO sample). Incubate at 37°C for 2 hours (static) to mimic organism exposure to antibiotics in patients. Take a 100 μL plate count sample (T2 sample) Add 5 mL of spiked blood + abx or blood only to a blood culture bottle (using a vial adapter) and begin incubation (remove the adapter) 5 mL of spiked blood + abx or blood only (from the same source) is processed with Momentum capture beads as follows: 1. Momentum capture beads diluted in detergent-free microbial binding buffer (DMBB) - 50 µL beads per cleanup: 700 µL DMBB Add 750 µL of diluted beads to a 2.50 mL conical centrifuge tube. Add 3.5 mL of migration medium to the tube Add 4.5 mL of spiked blood + abx or blood only to the tube. 5. Incubate in ITL TherMix (Integrated Technologies Ltd. Kent, UK) at 32.5°C / 30 min / 1000 rpm - ramp rate protocol 6. Magnetize on a V&P magnet (V&P Scientific Inc., CA, US) for 10 minutes, then remove the supernatant. 7. Transfer the beads to a 2 mL Eppendorf flip-top tube with 1 mL of detergent-free wash buffer (Thermo Fisher Scientific, Life Technologies Ltd. Paisley, UK) 8. Magnetize for 3 minutes, then remove the supernatant. 9. Resuspend the beads from the magnet in 1 mL of detergent-free wash buffer (DWB) and replace on the DynaMag-2 magnet 10. Magnetize for 3 minutes, then remove the supernatant. 11. Resuspend the beads in 1 mL DWB and add to a new blood culture bottle (using a vial adapter) and begin incubation (remove the adapter) Day 3: Record plate counts.

[0318] The "reversal" time for each bottle or bottle result is recorded at the end of the 5-day incubation. The "reversal" time is the time for the bottle to become positive as measured by an automated blood culture cabinet, and the time to positivity is recorded.

[0319] result: [Table 70]

[0320] comment: Although a difference of 2 to 4 hours was observed in the inversion time between horse and human blood, the final results were comparable.

[0321] Therefore, subsequent experiments can be performed using horse blood at this time when human blood is not available.

[0322] Example 19: Breakpoint Experiment Details the purpose: To determine starting points for antibiotic concentrations and microbial loads for subsequent experiments, a range of antibiotic concentrations was tested to find appropriate breakpoints for use in the experiments. A range of antibiotic concentrations was tested against a given microbial load to determine the concentration at which growth was inhibited but the microorganisms were not killed. This was measured by place counts at 0, 2.5, and 5 hour intervals.

[0323] Protocol: Day 1.1: Prepare an overnight culture of the test organism using 3 mL of blood / broth solution (1.5 mL defibrinated horse blood: 1.5 mL migration medium). Day 2.2: Prepare appropriate stock solutions by dissolving antibiotics to the required dilutions and prepare a dilution series in nuclease-free water to final concentrations ranging from 100 to 0.001 and 0 mg / L. 3. Dilute the overnight culture of the microorganism to the required point in the pre-warmed blood / broth solution. This may need to be determined for each organism / antibiotic combination, using 10 as a starting dilution. -4 is used (final concentration in the sample is 10 -5 (This becomes.) 4. Aliquot 980 μL of pre-warmed defibrinated horse blood into the required number of 2 mL flip-top tubes. 5. Add 10 μL of diluted antibiotic to the relevant tube and 10 μL of diluent to the final tube as a control. Mix. 6. Add 10 μL of the required dilution of the overnight culture of the microorganism. Mix. 7. Take a 100 μL sample from each tube and plate out on Columbia blood agar for time 0 counts. 8. Plate 100 μL of diluted overnight culture as growth / no growth control. 9. Samples are incubated at 37°C in a shaking incubator (450 rpm) and 100 μL is sampled at 2.5 and 5 hour intervals for plate counts. 10. Incubate the plate at 37°C overnight. 11. Day 3: Count plates and tabulate results. Determine the concentration of antibiotic that inhibits growth without completely killing the microorganisms.

[0324] The concentrations determined by this protocol were used in the following examples unless otherwise stated.

[0325] Example 20: Growth of organisms in the presence and absence of magnetic beads and / or antibiotics the purpose: The goal of this experiment is to evaluate whether the presence of magnetic beads allows organisms to grow faster because they bind to the beads and grow as if on a solid substrate. This experiment examines the relative growth rates of organisms in blood broth with and without antibiotics and with and without magnetic beads (no bead migration).

[0326] Protocol: Day 1: Prepare an overnight culture of the test organism in blood / broth medium (50:50 blood:broth) and incubate at 37° C. in a shaking incubator. Day 2: If necessary, prepare a 10-fold dilution series of the overnight culture in blood / broth and incubate at 37 °C for 2 h to grow.

[0327] In sample set 1, dilutions of test organisms in blood contain beads, and a replicate set of samples (set 2) does not contain beads. Neither set contains antibiotics. In sample set 3, the dilution of the test organism in blood contains antibiotics and beads, and a replicate set of samples (set 4) does not contain beads. Both sets contain antibiotics. Antibiotics are added to whole blood (defibrated horse blood) at the required level. Whole blood served as a control without antibiotics. Using 10 mL of pre-warmed whole blood per sample, add 10 µL of the test dilution of the organism per mL of blood. In a replicate set, add the relevant antimicrobial agent to each of three test tubes. 100 μL of these samples are plated for TO TVC. Five mL of the 10 mL sample is taken to create Set 1 and Set 3. Add 50 μL of raw magnetic beads per 5 mL of blood to sample tubes in Set 1 and Set 3, and no beads in Set 2 and Set 4. Add 5 mL of spiked blood + beads (set 1) and 5 mL of spiked blood / Abx + beads (set 3) to blood culture bottles (BioMerieux SA culture bottles) using vial adapters (West Pharmaceutical Services, Inc. PA, US). Next, 5 mL of spiked blood (from the same source), no-bead controls (Set 2 and Set 4) are added to the blood culture bottles. Remove the adapter and incubate the blood culture bottle in an automated blood culture cabinet (BacT / ALERT BioMerieux) for up to 5 days. Day 3: Count and record TVC plates.

[0328] The "reversal" time for each bottle or bottle result is recorded at the end of the 5-day incubation. The "reversal" time is the time for the bottle to become positive as measured by an automated blood culture cabinet, and the time to positivity is recorded.

[0329] result: [Table 71] comment: Incubation of the samples in the presence of magnetic beads did not aid or hinder the growth of the organisms.

[0330] Protocol for Biorescue Experiments (Examples 21 to 24) Day 1: Prepare an overnight culture of the test organism in blood / broth medium (50:50 blood:broth) and incubate at 37°C (or 30°C for Cryptococcus neoformans) in a shaking incubator. Day 2: In a clean room Prepare the appropriate volume of antibiotic to be used in the experiment. Prepare the stock solution using the formula 1000 / P x V x C = W. P = potency given by the manufacturer (µg / mg), V = volume required (mL), C = final concentration (multiple of 1000) of the solution (mg / L), and W = weight (mg) of antibiotic to be dissolved in volume V (mL). If necessary, prepare 10-fold serial dilutions of the overnight culture in blood / broth and incubate and grow for 2 hours at 37°C (or 30°C for Cryptococcus neoformans). Antibiotics are added to whole blood (defibrated horse blood) at the required levels. Whole blood as a control without antibiotics Add test dilution(s) of the organism to blood + antibiotic or blood only at a concentration of 10 µL / mL blood (prepared sufficiently for experimental requirements) Take a 100 μL plate count sample (T0 sample) Incubate at 37°C (or 30°C for Cryptococcus neoformans) for 2 hours (static). Take a 100 μL plate count sample (T2 sample) Add 5 mL of spiked blood + abx or blood only to a blood culture bottle (using a vial adapter) and begin incubation (remove the adapter) 5 mL of spiked blood + abx or blood only (from the same source) is processed with Momentum capture beads as follows: 1. Capture beads diluted to relevant capture: 50 µL beads: 700 µL capture reagent / cleanup Add 750 µL of diluted beads to a 2.50 mL Erlenmeyer centrifuge tube. Add 3.5 mL of migration medium to the tube Add 4.5 mL of spiked blood + abx or blood only to the tube. 5. Incubate in ITL TherMix (32.5°C / 30 min / 1000 rpm - ramped speed protocol) 6. Magnetize on a V&P magnet for 10 minutes, then remove the supernatant. 7. Resuspend the beads from the magnet in 1 mL of detergent-free wash buffer (DWB) and transfer to a 2 mL Eppendorf flip-top tube on the DynaMag-2 magnet 8. Magnetize for 3 minutes, then remove the supernatant. 9. Resuspend the beads from the magnet in 1 mL of detergent-free wash buffer (DWB) and replace on the DynaMag-2 magnet 10. Magnetize for 3 minutes, then remove the supernatant. 11. Resuspend the beads in 1 mL DWB and add to a new blood culture bottle (using a vial adapter) and begin incubation (remove the adapter) Day 3: Record plate counts.

[0331] The "reversal" time for each bottle or bottle result is recorded at the end of the 5-day incubation. The "reversal" time is the time for the bottle to become positive as measured by an automated blood culture cabinet, and the time to positivity is recorded.

[0332] Example 21: Comparison of Organism Rescue Using Detergent-Free Buffer or Hemolysis Capture Buffer the purpose: To compare detergent-free buffer or hemolysis capture buffer when performing organism rescue. Do different capture buffers affect successful organism rescue? Compare DMBB, E-Buff, and GMBB as capture reagents for recovery of E. coli and S. aureus, using DWB for all washes.

[0333] result: [Table 72]

[0334] comment: The turnover times between the different capture reagents for E. coli and S. aureus were very similar, indicating that this rescue method allows for recovery of the organism and can be performed with or without hemolytic agents.

[0335] Example 22: Application of biorescue to additional microorganisms and antimicrobial agents the purpose: To demonstrate that the rescue method is applicable to a wide variety of microorganisms and antimicrobial agents. Preparation of vancomycin: For vancomycin (Lot No. 058M4009V) with a potency of 1008 μg / mg, use the following formula to determine the weight needed to make 1 mL of a 10,000 mg / mL stock solution: Vancomycin stock solution was prepared using the formula 1000 / P x V x C = W. P = potency given by the manufacturer (µg / mg), V = volume required (mL), C = final concentration (multiple of 1000) of the solution (mg / L), and W = weight (mg) of antibiotic dissolved in volume V (mL). Therefore, 1000 / 1008×10mL×10=W 0.992×10×10=W For a 10,000 mg / mL stock solution, W = 99.2 mg in 10 mL of saline (0.9%).

[0336] Two different strains of S. aureus grew in all dilutions of vancomycin at all time points. Given that the drug is relatively slow acting and the length of the experiments needed to be kept within a reasonable time frame, it was decided to use a final concentration of 500 mg / L for subsequent experiments.

[0337] result: [Table 73]

[0338] comment: For the no antimicrobial control, blood cultures always reverted to positive before rescue procedures.

[0339] In standard SA bottles containing bacteria from blood with antimicrobial agents, rescue methods always reversed to positive before BC controls, which were negative on day 5. For Candida albicans, rescue methods reversed as much as 27 hours faster than BC controls, and for Cryptococcus neoformans, they reversed by over 13 hours.

[0340] Example 23: Comparison of biorescue using different magnetic beads type I the purpose: To perform rescue procedures using five different bead types: hydrophobic, aminated, carboxylated, SpeedBeads, and streptavidin-coated, and to compare the capture and recovery efficiencies. [Table 74]

[0341] result: [Table 75] [Table 76]

[0342] comment: All bead rescue samples reverted to positive within 24 hours (within 18 hours for S. aureus) compared with no-rescue blood culture controls, which were negative after 5 days.

[0343] This was true for all five beads tested for E. coli in blood containing 0.075 mg / L ciprofloxacin and S. aureus in blood containing 500 mg / L vancomycin.

[0344] Example 24: Comparison of biorescue using different magnetic beads type II the purpose: To perform rescue procedures using five additional bead types; citrate-capped, starch-capped, polyacrylamide, mannose-binding lectin, and polylysine, and compare the capture and recovery efficiencies. [Table 77]

[0345] result: [Table 78] [Table 79]

[0346] comment: All bead rescue samples reverted to positive for S. aureus within 17 hours compared with negative, non-rescue blood culture controls. This was true for all six beads tested for S. aureus in blood containing 500 mg / L vancomycin.

[0347] For E. coli, bead rescue samples reversed positive within 30 hours for all six beads tested in blood containing 0.075 mg / L ciprofloxacin. Blood culture controls (without bead rescue) were negative.

[0348] Example 25: Comparison of biorescue of unrecoverable organisms in FA plus BacT / ALERT blood culture bottles in the presence of antibiotics Protocol for replicating the work of Flayhart et al. and Chung et al. Day 1: Prepare an overnight culture of the test organism in blood / broth medium (50:50 blood:broth) and incubate at 37° C. in a shaking incubator. Day 2: In a clean room, prepare the appropriate volume of antibiotics to be used in the experiment. Prepare the stock solution using the formula 1000 / P x V x C = W. P = potency given by the manufacturer (µg / mg), V = volume required (mL), C = final concentration (multiple of 1000) of the solution (mg / L), and W = weight (mg) of antibiotic to be dissolved in volume V (mL). If necessary, prepare 10-fold serial dilutions of the overnight culture in blood / broth and incubate at 37 °C for 2 h to grow. Antibiotics are added to whole blood (defibrated horse blood) at the required levels. Whole blood as a control without antibiotics Add test dilution(s) of the organism to blood + antibiotic or blood only at a concentration of 10 µL / mL blood (prepared sufficiently for experimental requirements) Take a 100 μL plate count sample (T0 sample) Add 5 mL of spiked blood + abx or blood only to a blood culture bottle (BioMerieux FA + culture bottle) using the vial adapter and start incubation (remove the adapter) Process 5 mL of spiked blood + abx or blood only (from the same source) using Momentum capture beads as follows: 1. Dilute Momentum capture beads with the relevant capture reagent - 50 μL beads: 700 μL capture reagent / cleanup Add 750 µL of diluted beads to a 2.50 mL Erlenmeyer centrifuge tube. Add 3.5 mL of migration medium to the tube Add 4.5 mL of spiked blood + abx or blood only to the tube. 5. Incubate in ITL TherMix (32.5°C / 30 min / 1000 rpm - ramped speed protocol) 6. Magnetize on a V&P magnet for 10 minutes, then remove the supernatant. 7. Resuspend the beads from the magnet in 1 mL of detergent-free wash buffer (DWB) and transfer to a 2 mL Eppendorf flip-top tube on the DynaMag-2 magnet 8. Magnetize for 3 minutes, then remove the supernatant. 9. Resuspend the beads from the magnet in 1 mL of detergent-free wash buffer (DWB) and replace on the DynaMag-2 magnet 10. Magnetize for 3 minutes, then remove the supernatant. 11. Resuspend the beads in 1 mL DWB and add to an unused blood culture bottle (BioMerieux FA+ ​​culture bottle) using a vial adapter and start the incubation (remove the adapter) Day 3: Record plate counts.

[0349] The "reversal" time for each bottle or bottle result is recorded at the end of the 5-day incubation. The "reversal" time is the time for the bottle to become positive as measured by an automated blood culture cabinet, and the time to positivity is recorded.

[0350] Replication of the experiment by Flayhart et al., 2007 the purpose: To compare microbial capture and rescue from FA Plus bottles for ceftriaxone in Streptococcus pneumoniae. A replication study by Flayhart et al. (2007) tested antibiotic concentrations of 250, 125, and 94 mg / L, and also included concentrations of 50 and 10 mg / L.

[0351] result: In the table below, "Control" represents a sample processed using the FA Plus Bottle (also referred to as "BC" in the last column), and "Bead Rescue" represents a sample processed according to the method of the present invention. [Table 80]

[0352] comment: As reported by Flayhart et al., 2007, at high peak serum concentrations of antibiotic-blood (ceftriaxone), FA+ blood culture bottles are unable to recover Streptococcus pneumoniae. This data demonstrates that using the method of the present invention, Streptococcus pneumoniae can be recovered and successfully grown from blood containing high peak serum concentrations of antibiotic. This experiment demonstrates that the method of the present invention is an improvement over FA+ bottles as tested by Flayhart.

[0353] Replication of the experiment by Chung et al., 2019 the purpose: Compare organism capture and rescue from FA Plus bottles for the organisms in the table below containing each antibiotic. Additional antibiotic concentrations were tested as reported in Chung et al., 2019 (164 mg / L, 100 mg / L, and 49 mg / L for cefepime, cefotaxime, and meropenem, respectively). [Table 81]

[0354] result: In the table below, "Control" represents a sample processed using the FA Plus Bottle (also referred to as "BC" in the last column), and "Bead Rescue" represents a sample processed according to the method of the present invention. [Table 82]

[0355] comment: As reported by Chung et al., 2019, at the high peak serum concentrations of antibiotic-blood, FA+ blood culture bottles fail to recover microorganisms. This data demonstrates that using the method of the present invention, organisms can be recovered and successfully grown from blood containing antibiotics at high peak serum concentrations. This experiment demonstrates that the method of the present invention is an improvement over FA+ bottles as tested by Chung.

[0356] References: Flayhart D, Borek AP, Wakefield T, Dick J, Carroll KC. Comparison of BACTEC PLUS blood culture media to BacT / Alert FA blood culture media for detection of bacterial pathogens in samples containing therapeutic levels of antibiotics. J Clin Microbiol. 2007 Mar;45(3):816-21. doi: 10.1128 / JCM.02064-06. Epub 2006 Dec 13. PMID: 17166960; PMCID: PMC1829095. Chung Y, Kim IH, Han M, Kim HS, Kim HS, Song W, Kim JS. A comparative evaluation of BACT / ALERT FA PLUS and FN PLUS blood culture bottles and BD BACTEC Plus Aerobic and Anaerobic blood culture bottles for antimicrobial neutralization. Eur J Clin Microbiol Infect Dis. 2019 Dec;38(12):2229-2233. doi: 10.1007 / s--019-03663-3. Epub 2019 Aug 2. PMID: 31375943.

Claims

1. 1. A method for recovering viable microorganisms from a sample containing microbial cells and an antimicrobial agent, comprising: a) incubating the sample with coated particles to form particle-microorganism complexes; b) separating the particle-microorganism complexes from the antimicrobial agent, thereby recovering viable microorganisms from the sample; A method comprising:

2. 10. The method of claim 1, further comprising detecting and / or characterizing the recovered viable microorganisms.

3. 1. A method for detecting the absence or presence of viable microorganisms in a sample suspected of containing a microorganism, the method comprising: a) incubating the sample with coated particles to form particle-microorganism complexes if microorganisms are present in the sample; b) separating the particle-microorganism complexes from the antimicrobial agent, thereby recovering viable microorganisms from the sample; c) verifying the absence or presence of the recovered microorganisms; A method comprising:

4. The method according to any one of claims 1 to 3, further comprising the step of incubating and / or culturing the viable microorganisms recovered in step b).

5. 1. A method for incubating and / or culturing viable microorganisms recovered from a sample containing microbial cells and an antimicrobial agent, comprising: a) incubating the sample with coated particles to form particle-microorganism complexes; b) separating the particle-microorganism complexes from the antimicrobial agent, thereby recovering viable microorganisms from the sample; c) incubating and / or culturing the recovered viable microorganisms; A method comprising:

6. 10. A method for detecting the absence or presence of a microbial infection in a subject, comprising the step of performing the method of claim 5 on a sample from the subject, optionally wherein the subject has been treated with an antimicrobial agent.

7. 7. The method of claim 6, further comprising the step of characterizing the microorganism involved in the infection.

8. 8. The method of any one of claims 1 to 7, wherein the sample also contains non-microbial cells, and step b) separates the particle-microorganism complexes from the antimicrobial agent and the non-microbial cells; optionally, the non-microbial cells comprise blood cells.

9. The method according to any one of claims 1 to 8, wherein step b) is carried out in the absence of a surfactant.

10. 9. The method according to claim 1, wherein step b) is carried out in the presence of sodium polyanethole sulfonate and / or a reagent that selectively lyses non-microbial cells in the sample while preserving intact microorganisms present in the sample.

11. 11. The method of any one of claims 1 to 10, wherein step b) comprises washing the separated particle-microorganism complexes, and optionally the separated particle-microorganism complexes are washed with a solution that does not contain a surfactant.

12. The method according to any one of claims 1 to 11, wherein step b) comprises using a magnetic field or centrifugation.

13. a) a sample containing an antimicrobial agent and viable microorganisms; b) coated particles capable of forming complexes with viable microorganisms in the sample; A composition comprising:

14. A composition comprising a sample containing an antimicrobial agent and viable microorganisms complexed with coated particles.

15. 15. The composition of claim 13 or claim 14, wherein the sample containing viable microbial cells further comprises non-microbial cells, optionally wherein the non-microbial cells comprise blood cells.

16. a) a container containing coated particles capable of forming complexes with viable microorganisms; b) a container containing a medium suitable for incubating and / or culturing the viable microorganisms recovered using the coated particles, the medium being free of antimicrobial agents and / or agents capable of binding to antimicrobial agents; and / or c) a container containing a wash buffer for washing the coated particles recovered from the sample, the wash buffer not lysing viable microorganisms; A kit for carrying out the method according to any one of claims 1 to 12, comprising:

17. 17. The kit of claim 16, comprising components a), b) and c) (in separate containers).

18. The wash buffer a) surfactant-free; b) containing Tris and / or sodium chloride; and / or c) does not lyse non-microbial cells; 18. The kit of claim 17.

19. a) a nucleic acid molecule that acts as a substrate for the nucleic acid modifying activity of viable microorganisms, thus allowing the detection of whether microorganisms are present in a sample; and / or b) primers and / or probes that specifically hybridize to nucleic acids from viable microorganisms, thus allowing the characterization of the microorganisms in the sample; The kit of any one of claims 16 to 18, further comprising:

20. The method of any one of claims 1 to 12, the composition of any one of claims 13 to 15, or the kit of any one of claims 16 to 19, wherein the microorganism is a bacterium or a fungus.

21. 21. The method of any one of claims 1 to 12 and 20, the composition of any one of claims 13 to 15 and 20, or the kit of any one of claims 16 to 20, wherein the antimicrobial agent is an antibiotic or an antifungal agent.

22. The method of any one of claims 1 to 12, 20 and 21, the composition of any one of claims 13 to 15, 20 and 21, or the kit of any one of claims 16 to 21, wherein the sample is a clinical sample taken from a subject undergoing or having undergone treatment with the antibacterial agent.

23. The method of any one of claims 1 to 12 and 19 to 22, the composition of any one of claims 13 to 15 and 19 to 22, or the kit of any one of claims 16 to 22, wherein the sample is selected from blood, cerebrospinal fluid (CSF), synovial fluid, urine, and bronchoalveolar lavage (BAL).

24. 24. The method of any one of claims 1 to 12 and 19 to 23, the composition of any one of claims 13 to 15 and 19 to 23, or the kit composition of any one of claims 16 to 23, wherein the sample comprises blood or a blood culture sample, optionally wherein the sample comprises whole blood.

25. The method of any one of claims 1 to 12 and 19 to 24, the composition of any one of claims 13 to 15 and 19 to 24, or the kit of any one of claims 16 to 24, wherein the coated particles are magnetic.

26. 26. The method of any one of claims 1 to 12 and 19 to 25, the composition of any one of claims 13 to 15 and 19 to 25, or the kit of any one of claims 16 to 25, wherein the coated particles comprise a polymeric surface, and optionally the polymeric surface comprises a carbon-based polymer.

27. The coated particles have on their outer surface: i) Carboxylic acid group ii) amino group iii) a hydrophobic group, and iv) Streptavidin The method of any one of claims 1 to 12 and 19 to 25, the composition of any one of claims 13 to 15 and 19 to 25, or the kit of any one of claims 16 to 25, comprising any one or more of:

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