SPS mitigation

Abstract

This disclosure relates to removal of a first polyanionic polymer from a clinical sample by addition of a second polyanionic polymer to the clinical sample, which second polyanionic polymer may be the same or different from the first polyanionic polymer.

Description

SPS MITIGATIONRELATED APPLICATIONS

[0001] This Application claims priority to U.S. Provisional Application Serial No. 63 / 601,959, entitled “SPS MITIGATION” filed on November 22, 2023, the entire contents of which are incorporated herein by reference.GOVERNMENT SUPPORT

[0002] This invention was made with U.S. Government support under Agreement Number 75A50122C00028, awarded by the U.S. Department of Health and Human Services. The U.S. Government has certain rights in the invention.BACKGROUND

[0003] Clinical sample processing requires selective removal of subject (e.g., human) cells from microbial cells from one or more microbial species. Microbial cells are preserved intact by the presence of one or more polyanionic polymers in a clinical sample.SUMMARY

[0004] Although polyanionic polymers can be useful to preserve microbial cells, they can also inhibit downstream clinical sample processing, including interfering with nucleic acid amplification.

[0005] The disclosure relates to the unexpected discovery that polyanionic polymers can effectively be removed from whole blood clinical samples by adding a greater amount of polyanionic polymers (e.g., sodium polyanethol sulfonate (SPS)) to the samples. This method also permits intact microbe preservation and subsequent molecular diagnostics of the clinical sample. Utilizing this method, human patient-derived clinical samples can be rapidly processed with SPS to identify microbes that are present.

[0006] Thus, the present disclosure provides methods for removing a polyanionic polymer from a clinical sample. In some embodiments, the method comprises: (i) obtaining a clinical sample from a subject comprising microbial species in a container comprising between 0.01% and 1.00% of a first polyanionic polymer, (ii) adding between 0.1% and 10%of a second polyanionic polymer to the clinical sample, (iii) removing from the clinical sample from step (ii) a first fraction comprising the first polyanionic polymer and the second polyanionic polymer.

[0007] The present disclosure provides methods for amplifying microbial DNA in a clinical sample collected from blood. In some embodiments, the method comprises: (i) obtaining a clinical sample from a subject comprising microbial species in a container comprising between 0.01% and 1.00% of a first polyanionic polymer, (ii) adding between 0.1% and 10% of a second polyanionic polymer to the clinical sample, (iii) removing from the clinical sample from step (ii) a first fraction comprising the one or more microbial species and cells from the subject and a second fraction comprising the first polyanionic polymer and the second polyanionic polymer, (iv) lysing the one or more microbial species and cells from the subject from the first fraction, and (vi) amplifying microbial DNA from the lysed microbial species.

[0008] The present disclosure provides methods for amplifying microbial DNA in a clinical sample collected from blood. In some embodiments, the method comprises: (i) obtaining a clinical sample from a subject comprising microbial species in a container comprising between 0.01% and 1.00% of a first polyanionic polymer, (ii) adding between 0.1% and 10% of a second polyanionic polymer to the clinical sample, (iii) removing from the clinical sample from step (ii) a first fraction comprising the one or more microbial species and cells from the subject and a second fraction comprising the first polyanionic polymer and the second polyanionic polymer, (iv) lysing the one or more microbial species and cells from the subject from the first fraction, (v) amplifying microbial DNA from the lysed microbial species, and (vi) identifying microbial species from the amplified microbial DNA.

[0009] The present disclosure provides methods for removing a first polyanionic polymer (e.g., sodium polyanethol sulfonate (SPS)) in a clinical sample containing nucleic acids. In some embodiments, the method comprises: (a) adding a second polyanionic polymer to the clinical sample, (b) solubilizing polyanionic polymer-protein precipitates, and (c) removing the soluble fraction comprising the first polyanionic polymer and the second polyanionic polymer. In some embodiments, the first polyanionic polymer and the second polyanionic polymer are the same. In some embodiments, the first polyanionic polymer and the second polyanionic polymer are different.

[0010] In some aspects, the present disclosure provides methods for reducing the amount of a first polyanionic polymer (e.g., sodium polyanethol sulfonate (SPS)) in a clinicalsample containing nucleic acids. In some embodiments, the method comprises: (a) adding a second polyanionic polymer to the clinical sample, (b) solubilizing polyanionic polymer- protein precipitates, and (c) removing the soluble fraction comprising the first polyanionic polymer and the second polyanionic polymer.

[0011] In some embodiments, the amplification is whole genome amplification. In some embodiments, adding a polyanionic polymer nucleic acid amplification inhibitor increases the amount of amplified microbial DNA between 2-fold and 100-fold, compared to control. In some embodiments, the separating is by centrifugation.

[0012] In some embodiments, a first polyanionic polymer is sodium polyanethol sulfonate (SPS). In some embodiments, a second polyanionic polymer is SPS. In some embodiments, a first polyanionic polymer and a second polyanionic polymer is SPS. In some embodiments, between 0.1% and 10% of a second polyanionic polymer is added to a clinical sample comprising blood.

[0013] In some embodiments, the subject is a human. In some embodiments, the microbe is a pathogenic microbe.BRIEF DESCRIPTION OF DRAWINGS

[0014] The accompanying drawings are not intended to be drawn to scale. The drawings are illustrative only and are not required for enablement of the disclosure. For purposes of clarity, not every component may be labeled in every drawing. In the drawings:

[0015] Figures 1A-1B illustrate that SPS forms large macromolecular aggregates with lysozyme. SPS-lysozyme aggregates form a pellet in the bottom of a tube following centrifugation (Fig. 1A). Addition of high concentration SPS prevents formation of SPS- lysozyme aggregates in a pellet following centrifugation (Fig. IB).

[0016] Figures 2A-2C illustrate that high concentration SPS improves microbial DNA amplification and sequencing outputs from whole blood. Addition of 0.5%, 1% or 5% SPS to whole blood samples collected in SPS vacutainers increased microbial DNA amplification by 100-fold (Fig. 2A), and increased post-quality control (QC) total number of megabases (MBs) (Fig. 2B) relative to control whole blood samples with 0% SPS added.Bacterial whole genome coverage values were also higher in samples treated with 0.5%, 1% or 5% SPS, as compared to no-SPS control (Fig. 2C).

[0017] Figure 3 demonstrates that SPS mitigation process significantly increases microbial DNA recovery when using SPS containing vacutainers, compared to otheranticoagulant-containing tubes, such as ACD-A, EDTA or heparin. The dotted line illustrates the normalized bacterial genome ID value.DETAILED DESCRIPTION

[0018] Clinical sample processing for microbial identification involves collecting a clinical sample from a subject, processing the clinical sample, and identifying the microbe(s) present in the clinical sample. This clinical sample processing relies on preserving intact microbial cells to enable their processing and identification. One or more polyanionic polymers (PPs) may be added to clinical samples to preserve intact microbial (e.g., bacterial) cells by inhibiting protein degradation (e.g., by the complement pathway). However, these PPs may inhibit downstream molecular diagnostic reactions (e.g., amplification, sequencing) and need to either be removed from or mitigated in clinical samples in order to perform molecular diagnostic reactions utilizing the clinical samples.

[0019] The present disclosure is based on the unexpected finding that high concentrations of one or more PPs (e.g., SPS) added to a clinical sample (e.g., whole blood sample) already comprising PPs is effective in mitigating the PPs. Without wishing to be bound by theory, this appears to be due to the added PPs disrupting macromolecular precipitates. Disrupting macromolecular precipitates increases the efficiency of downstream molecular diagnostic reactions. In some embodiments, the present disclosure therefore provides a method for mitigating PPs in a clinical sample to enable intact microbe preservation and subsequent molecular diagnostics of the clinical sample. This method enables rapid processing of a clinical sample with PPs to identify the microbes present.

[0020] The present disclosure therefore advances the field of clinical sample processing and molecular diagnostics by providing methods for maintaining intact microbial cells that can be processed from collection through molecular diagnostic reactions without inhibition by PPs. Other methods currently in the field rely on (1) anticoagulants such as EDTA, which does not prevent all complement pathway activity and microbial cell lysis: and / or (2) organic extractions of PPs that cause microbial cell lysis.Polyanionic polymers

[0021] The present disclosure provides methods for removing a polyanionic polymer (PP) from a clinical sample by adding more additional polyanionic polymers (PPs) to the clinical sample. As used herein, a “polyanionic polymer” is a non-proteinaceous substancewith a molecular structure consisting of multiple similar chemical units bonded together and comprising multiple negatively-charged groups, and that has an average molecular weight of at least 500 daltons (500 Da). Non-limiting examples of polyanionic polymers include: SPS, heparin, hyaluronate, dermatan sulfate polyanion, and chondroitin D-glucuronate anion. In some embodiments, a polyanionic polymer is SPS.

[0022] PPs may inhibit nucleic acid (e.g., DNA, RNA) amplification in a clinical sample. Without wishing to be bound by theory, PPs in a clinical sample may form macromolecular aggregates containing, e.g., PPs, proteins, nucleic acids (e.g., microbial, human). These macromolecular aggregates may inhibit certain molecular processes due to physical sequestration of proteins and / or nucleic acids, chelation of ions, or any other mechanism for inhibiting molecular processes. In some embodiments, macromolecular aggregates containing PPs inhibit nucleic acid amplification. It is therefore advantageous to have PPs in a clinical sample prior to nucleic acid amplification (e.g., obtaining the clinical sample, separating human cells from microbial cells, lysing human cells, lysing microbial cells, or some combination thereof), and to remove the PPs prior to nucleic acid amplification. Nucleic acid amplification may be by any method provided herein or known to a person skilled in the art.

[0023] PPs may be completely or partially removed from a clinical sample. In some embodiments, 10% - 100% of PPs are removed from a clinical sample. In some embodiments, 15% - 95%, 20% - 90%, 25% - 85%, 30% - 80%, 35% - 75%, 40% - 70%, 45% - 65%, or 50% - 60% of PPs are removed from a clinical sample. In some embodiments, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, 30%, 32%, 34%, 36%, 38%, 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, or 100% of PPs are removed from a clinical sample by a method provided herein.

[0024] PPs in a clinical sample may inhibit nucleic acid amplification in a clinical sample by 10% - 100%, 15% - 95%, 20% - 90%, 25% - 85%, 30% - 80%, 35% - 75%, 40%, - 70%, 45% - 65%, or 50 - 55% compared to a control. In some embodiments, PPs may inhibit nucleic acid amplification in a clinical sample by 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, 30%, 32%, 34%, 36%, 38%, 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 100% or more compared to a reference sample, such as a clinical sample that never contained a PP, or a clinical sample that has had PPs removed.

[0025] Clinical sample containers may include polyanionic polymers (PPs) to protect intact microbial cells during clinical sample processing. For example, commonly used sample collection containers, sample culture bottles, plasma tubes, and blood culture media may include heparin (e.g., Cat. Nos. 364960, 366667, 367871, 367878, 367884, 367886, 367960, 367961, 367962, and 367964 Vacutainer® collection tubes, BD Biosciences, Franklin Lakes, NJ), sodium polyanethol sulfonate (SPS, e.g., Cat. No. 364960 Vacutainer® collection tubes, Cat. Nos. 442022 and 442023, BACTECTM PLUS media, BD Biosciences, Franklin Lakes, NJ) or potassium EDTA (e.g., Cat. Nos. 367842, 367899 and 368589, Vacutainer® Plus Plastic K2EDTA Tubes, BD Biosciences, Franklin Lakes, NJ).

[0026] In some embodiments, a PP is SPS. SPS is an anticoagulant that prevents microbial (e.g., bacterial) lysis by innate cellular and humoral factors. SPS inhibits the mammalian (e.g., human) complement system to inhibit microbial (e.g., bacterial) phagocytosis. Commercial concentrations of SPS range from 0.25 mg / mL to 0.5 mg / mL.

[0027] In some embodiments, a clinical sample container comprises 0.05 mg / mL - 5.0 mg / mL of a first PP. In some embodiments, a clinical sample container comprises 0.10 mg / mL - 4.9 mg / mL, 0.20 mg / mL - 4.8 mg / mL, 0.30 mg / mL - 4.7 mg / mL, 0.40 mg / mL - 4.6 mg / mL, 0.50 mg / mL - 4.5 mg / mL, 0.6 mg / mL - 4.4 mg / mL, 0.7 mg / mL - 4.3 mg / mL, 0.8 mg / mL - 4.2 mg / mL, 0.9 mg / mL - 4.1 mg / mL, 1.0 mg / mL, 4.0 mg / mL, 1.1 mg / mL - 3.9 mg / mL, 1.2 mg / mL - 3.8 mg / mL, 1.3 mg / mL - 3.7 mg / mL, 1.4 mg / mL - 3.6 mg / mL, 1.5 mg / mL - 3.5 mg / mL, 1.6 mg / mL - 3.4 mg / mL, 1.7 mg / mL - 3.3 mg / mL, 1.8 mg / mL - 3.2 mg / mL, 1.9 mg / mL - 3.1 mg / mL, 2.0 mg / mL - 3.0 mg / mL, 2.1 mg / mL - 2.9 mg / mL, 2.2 mg / mL - 2.8 mg / mL, 2.3 mg / mL - 2.7 mg / mL, 2.4 mg / mL - 2.6 mg / mL of a first PP. In some embodiments, a clinical sample container comprises 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 1.1 mg / mL, 1.2 mg / mL, 1.3 mg / mL, 1.4 mg / mL, 1.5 mg / mL, 1.6 mg / mL, 1.7 mg / mL, 1.8 mg / mL, 1.9 mg / mL, 2.0 mg / mL, 2.1 mg / mL, 2.2 mg / mL, 2.3 mg / mL, 2.4 mg / mL, 2.5 mg / mL, 2.6 mg / mL, 2.7 mg / mL, 2.8 mg / mL, 2.9 mg / mL, 3.0 mg / mL, 3.1 mg / mL, 3.2 mg / mL, 3.3 mg / mL, 3.4 mg / mL, 3.5 mg / mL, 3.6 mg / mL, 3.7 mg / mL, 3.8 mg / mL, 3.9 mg / mL, 4.0 mg / mL, 4.1 mg / mL, 4.2 mg / mL, 4.3 mg / mL, 4.4 mg / mL, 4.5 mg / mL, 4.6 mg / mL, 4.7 mg / mL, 4.8 mg / mL, 4.9 mg / mL, or 5.0 mg / mL or more of a first PP.

[0028] In some aspects, a clinical sample container comprises 0.01% - 1.00% concentration of a first PP after a clinical sample (e.g., whole blood) is added. In some embodiments, a clinical sample container comprises 0.05% - 0.95%, 0.10% - 0.90%, 0.15% - 0.85%, 0.20% - 0.80%, 0.25% - 0.75%, 0.30% - 0.70%, 0.35% - 0.65%, 0.40% - 0.60%,0.45% - 0.55% of a first PP after a clinical sample is added. In some embodiments, a clinical sample container comprises 0.01%, 0.03%, 0.05%, 0.07%, 0.09%, 0.11%, 0.13%, 0.15%, 0.17%, 0.19%, 0.21%, 0.23%, 0.25%, 0.27%, 0.29%, 0.31%, 0.33%, 0.35%, 0.37%, 0.39%,0.41%, 0.43%, 0.45%, 0.47%, 0.49%, 0.51%, 0.53%, 0.55%, 0.57%, 0.59%, 0.61%, 0.63%,0.65%, 0.67%, 0.69%, 0.71%, 0.73%, 0.75%, 0.77%, 0.79%, 0.81%, 0.83%, 0.85%, 0.87%,0.89%, 0.91%, 0.93%, 0.95%, 0.97%, 0.99% or more of a first PP after a first clinical sample is added.

[0029] PPs may be added to a clinical sample already containing one or more PPs in a single addition or in multiple (e.g., 2 or more) additions. These multiple PPs additions may be simultaneous or non-simultaneous. Simultaneous means that multiple PP additions occur at exactly the same time. Non-simultaneous means that multiple PP additions occur at different times that are separated by e.g., seconds, minutes, hours, or more. Multiple additions that are simultaneously added may mean that one or more PPs are encapsulated in separate devices (e.g., tube, pipette tip, etc.), and their addition occurs at the same time. Multiple additions that are non-simultaneous may be that one or more PPs are encapsulated in separate devices and their addition does not occur at the same time.

[0030] PPs added to a clinical sample may be the same PP as is already in the clinical sample or a different PP that is already in the clinical sample. In some embodiments, the same PP as is already in a clinical sample is added to a clinical sample. In some embodiments, a different PP is added to a clinical sample compared to the PP already in the clinical sample. In some embodiments, a clinical sample container comprises a first PP and a second PP is added to the clinical sample. The first PP and the second PP may be the same PP or different PPs. In some embodiments, the first PP and the second PP are the same. In some embodiments, the first PP and the second PP are different.

[0031] To a clinical sample comprising the first PP may be added up 1-10 different PPs. In some embodiments, 1-10 different PPs, 2-9 different PPs, 3-8 different PPs, 4-7 different PPs, or 5-6 different PPs may be added to a clinical sample comprising the first PP. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more different PPs are added to a clinical sample comprising a first PP.

[0032] In some embodiments, 1-10 different PPs are added to a clinical sample at an aggregate concentration of 0.05 mg / mL - 5.0 mg / mL of 1-10 different PPs. In some embodiments, 1-10 different PPs are added to a clinical sample at an aggregate concentration of 0.10 mg / mL - 4.9 mg / mL, 0.20 mg / mL - 4.8 mg / mL, 0.30 mg / mL - 4.7 mg / mL, 0.40 mg / mL - 4.6 mg / mL, 0.50 mg / mL - 4.5 mg / mL, 0.6 mg / mL - 4.4 mg / mL, 0.7 mg / mL - 4.3mg / mL, 0.8 mg / mL - 4.2 mg / mL, 0.9 mg / mL - 4.1 mg / mL, 1.0 mg / mL, 4.0 mg / mL, 1.1 mg / mL - 3.9 mg / mL, 1.2 mg / mL - 3.8 mg / mL, 1.3 mg / mL - 3.7 mg / mL, 1.4 mg / mL - 3.6 mg / mL, 1.5 mg / mL - 3.5 mg / mL, 1.6 mg / mL - 3.4 mg / mL, 1.7 mg / mL - 3.3 mg / mL, 1.8 mg / mL - 3.2 mg / mL, 1.9 mg / mL - 3.1 mg / mL, 2.0 mg / mL - 3.0 mg / mL, 2.1 mg / mL - 2.9 mg / mL, 2.2 mg / mL - 2.8 mg / mL, 2.3 mg / mL - 2.7 mg / mL, 2.4 mg / mL - 2.6 mg / mL of a first PP. In some embodiments, a clinical sample container comprises 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 1.1 mg / mL, 1.2 mg / mL, 1.3 mg / mL, 1.4 mg / mL, 1.5 mg / mL, 1.6 mg / mL, 1.7 mg / mL, 1.8 mg / mL, 1.9 mg / mL, 2.0 mg / mL, 2.1 mg / mL, 2.2 mg / mL, 2.3 mg / mL, 2.4 mg / mL, 2.5 mg / mL, 2.6 mg / mL, 2.7 mg / mL, 2.8 mg / mL, 2.9 mg / mL, 3.0 mg / mL, 3.1 mg / mL, 3.2 mg / mL, 3.3 mg / mL, 3.4 mg / mL, 3.5 mg / mL, 3.6 mg / mL, 3.7 mg / mL, 3.8 mg / mL, 3.9 mg / mL, 4.0 mg / mL, 4.1 mg / mL, 4.2 mg / mL, 4.3 mg / mL, 4.4 mg / mL, 4.5 mg / mL, 4.6 mg / mL, 4.7 mg / mL, 4.8 mg / mL, 4.9 mg / mL, or 5.0 mg / mL of 1-10 different PPs.

[0033] In some embodiments, 1-10 different PPs are added to a clinical sample in 0.1% - 50% of the total concentration of the first PP. In some embodiments, 1-10 different PPs are added to a clinical sample in 0.1% - 49%, 0.5% - 48%, 1% - 47%, 2% - 46%, 3% - 45%, 4% - 44%, 5% - 43%, 6% - 42%, 7% - 41%, 8% - 40%, 9% - 39%, 10% - 38%, 11% - 37%, 12% - 36%, 13% - 35%, 14% - 34%, 15% - 33%, 16% - 32%, 17% - 31%, 18% - 30%, 19% - 29%, 20% - 28%, 21% - 27%, 22% - 26%, 23% - 25% of the total concentration of the first PP. In some embodiments, 1-10 different PPs are added to a clinical sample in 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50% or more of the total concentration.

[0034] In some embodiments, 1-10 different PPs are added to a clinical sample in 1% - 500% of the total volume of the first PP. In some embodiments, 1-10 different PPs are added to a clinical sample in 5% - 495%, 10% - 490%, 20% - 480%, 30% - 470%, 40% - 460%, 50% - 450%, 60% - 440%, 70% - 430%, 80% - 420%, 90% - 410%, 100% - 400%, 110% - 390%, 120% - 380%, 130% - 370%, 140% - 360%, 150% - 350%, 160% - 340%, 170% - 330%, 180% - 320%, 190% - 310%, 200% - 300%, 210% - 290%, 220% - 280%, 230% - 270%, 240% - 260% of the total volume of the first PP. In some embodiments, 1-10 different PPs are added to a clinical sample in 1%, 5,%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%,330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500% or more of the total volume of the first PP.

[0035] In some embodiments, 1-10 different PPs are added to a clinical sample in 1%- 500% of the total concentration of the first PP. In some embodiments, 1-10 different PPs are added to a clinical sample in 5% - 495%, 10% - 490%, 20% - 480%, 30% - 470%, 40% - 460%, 50% - 450%, 60% - 440%, 70% - 430%, 80% - 420%, 90% - 410%, 100% - 400%, 110% - 390%, 120% - 380%, 130% - 370%, 140% - 360%, 150% - 350%, 160% - 340%, 170% - 330%, 180% - 320%, 190% - 310%, 200% - 300%, 210% - 290%, 220% - 280%, 230% - 270%, 240% - 260% of the total concentration of the first PP. In some embodiments, 1-10 different PPs are added to a clinical sample in 1%, 5,%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500% or more of the total concentration of the first PP.

[0036] In some embodiments, 1-10 different PPs are added to a clinical sample in 1%- 500% of the total volume of a clinical sample comprising a first PP. In some embodiments, 1-10 different PPs are added to a clinical sample in 5% - 495%, 10% - 490%, 20% - 480%, 30% - 470%, 40% - 460%, 50% - 450%, 60% - 440%, 70% - 430%, 80% - 420%, 90% - 410%, 100% - 400%, 110% - 390%, 120% - 380%, 130% - 370%, 140% - 360%, 150% - 350%, 160% - 340%, 170% - 330%, 180% - 320%, 190% - 310%, 200% - 300%, 210% - 290%, 220% - 280%, 230% - 270%, 240% - 260% of the total volume of a clinical sample comprising a first PP. In some embodiments, 1-10 different PPs are added to a clinical sample in 1%, 5,%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500% or more of the total volume of a clinical sample comprising a first PP.

[0037] Adding 1-10 different PPs to a clinical sample comprising a first PP inhibits or prevents the formation of macromolecular aggregates comprising the first PP (Figures 1A, IB). By adding 1-10 different PPs that bind to a first PP, the PPs can be removed from the clinical sample because the PPs are inhibited or prevented from forming macromolecular aggregates. This removal may be by any method known in the art. Non-limiting methods of removing PPs from a clinical sample include: splitting a clinical sample into multiple fractions, binding of the PPs with a protein (e.g., bovine serum albumin, human serumalbumin, etc.), and affinity-based separation (e.g., antibody-based binding, magnetic bead binding).

[0038] In some embodiments, removal of a PP from a clinical sample comprises removing a fraction comprising a first PP and a second PP from a clinical sample. The clinical sample may comprise one or more microbial species. In some embodiments, a clinical sample comprising a first PP and 1-10 different PPs is separated into multiple fractions. This separation may be by any method known in the art including, but not limited to: velocity sedimentation and size exclusion chromatography. In some embodiments, a clinical sample comprising a first PP and 1-10 different PPs is separated into multiple fractions by velocity sedimentation.

[0039] In some embodiments, a clinical sample is separated into a first fraction comprising one or more microbial species and cells from a subject and a 2-10 additional fractions comprising the 1-10 different PPs.

[0040] In some embodiments, a clinical sample is split into at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or more fractions. In some embodiments, a clinical sample is split into 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4,3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, 6-7, 6-8, 6- 9, 6-10, 7-8, 7-9, 7-10, 8-9, 8-10, or 9-10 or more fractions.

[0041] In some embodiments, a clinical sample is split into at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or more fractions that are equal in weight and / or volume. In some embodiments, a clinical sample is split into at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or more fractions. In some embodiments, a clinical sample is split into 2-3, 2-4, 2-5, 2-6, 2-7, 2- 8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, 6-7, 6-8, 6-9, 6-10, 7-8, 7-9, 7-10, 8-9, 8-10, or 9-10 fractions that are equal in weight and / or volume.

[0042] In some embodiments, a clinical sample is split into at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or more fractions that are unequal in weight and / or volume. In some embodiments, a clinical sample is split into 2, 3, 4, 5, 6, 7, 8, 9, 10, or more fractions. In some embodiments, a clinical sample is split into 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9,4-10, 5-6, 5-7, 5-8, 5-9, 5-10, 6-7, 6-8, 6-9, 6-10, 7-8, 7-9, 7-10, 8-9, 8-10, or 9-10 fractions that are unequal in weight and / or volume. In some embodiments, a clinical sample is splitinto at least a first fraction (e.g., fraction 1) comprising one or more microbial species and cells from the subject and a second fraction (e.g., fraction 2) comprising 1-10 PPs.

[0043] In some embodiments, the present disclosure provides a method for removing a polyanionic polymer from a clinical sample, the method comprising: (i) obtaining a clinical sample from a subject comprising one or more microbial species in a container comprising 0.01% - 1.00% of a first polyanionic polymer; (ii) adding between 0.1% - 10% of a second polyanionic polymer to the clinical sample; (iii) removing from the clinical sample from step (ii) a first fraction comprising the first polyanionic polymer and the second polyanionic polymer.

[0044] In some embodiments, the present disclosure provides a method for removing a polyanionic polymer from a clinical sample, the method comprising: (i) obtaining a clinical sample from a subject comprising one or more microbial species in a container comprising 0.01% - 1.00% of a first polyanionic polymer nucleic acid amplification inhibitor; (ii) adding between 0.1% - 10% of a second polyanionic polymer nucleic acid amplification inhibitor to the clinical sample; (iii) separating the clinical sample from step (ii) into a first fraction comprising the one or more microbial species and cells from the subject and a second fraction comprising the first polyanionic polymer nucleic acid amplification inhibitor and the second polyanionic polymer nucleic acid inhibitor; and (iv) removing the second fraction from the clinical sample in step (iii).

[0045] The skilled person will appreciate that in some instances, polycationic and / or zwitterionic polymers may also be removed from a clinical sample according to any method provided herein. Polycationic polymers are compounds composed of monomers comprising one or more positive charges. A poly cationic polymer may be any polycationic polymer known in the art in clinical sample processing. Non-limiting examples of polycationic polymers include: polyamidoamine, polyethylenimine (PEI), mannosylated PEI, poly(L- lysine), and poly(P-amino ester). Zwitterionic polymers are compounds composed of monomers comprising one or more positive charges and one or more negative charges, wherein the polymer has an overall net neutral charge. A zwitterionic polymer may be any zwitterionic polymer known in the art in clinical sample processing. Non-limiting examples of zwitterionic polymers include: polycarboxybetaine, polysulfobetaine, polycysteine, phosphatidylcholine, poly(acrylamide-co-(3-(l-(4-vinylbenzyl)-lH-benzo[d]imidazole-3- ium-3-yl)propane-l -sulfonate)), and poly ampholytes.Clinical Sample ProcessingSeparating Clinical Sample

[0046] In some embodiments, methods provided herein include separating a clinical sample comprising 1-10 PPs to remove the 1-10 PPs from microbial cells and human cells into a fraction(s) comprising one or more microbial cells and subject cells and a fraction(s) comprising the 1-10 PPs. The fraction(s) comprising microbial cells and subject cells is removed from a fraction comprising the 1-10 PPs, or a fraction(s) comprising the 1-10 PPs is removed from a fraction(s) comprising microbial cells and subject cells. The term “separating”, as used herein, refers to the process of physically separating two or more substances (e.g., microbial cells and subject cells, 1-10 PPs). In some embodiments, separating may include centrifugation (e.g., velocity sedimentation), separating supernatant (e.g., from a pellet), and moving the supernatant to a separate tube (e.g., from the pellet), filtration, nucleic acid (e.g., DNA or RNA) isolation, or any combination thereof.

[0047] In some embodiments, separating may include one or more centrifugation steps, such as velocity sedimentation. Velocity sedimentation, also known as rate-zonal centrifugation, is a technique used to separate particles in a solution based on their size and shape, where larger molecules accumulate in a pellet at the bottom of the container (e.g., tube) being centrifuged and smaller molecules accumulate in the supernatant above the pellet. This method can be employed to fractionate macromolecules (e.g., proteins, nucleic acids, or subcellular organelles) according to their sedimentation rates in a centrifugal field. In the present disclosure, centrifugation may be used to compact human cells and microbial cells in the bottom of a container (e.g., tube). In some embodiments, where centrifugation is used to compact human cells and microbial cells at the bottom of a tube, a method of the present disclosure may further comprise removing supernatant from above the compacted human cells (e.g., comprising 1-10 PPs and placing it into a separate tube.

[0048] In some aspects, methods provided herein include removing a fraction from a clinical sample. As used herein, “removing” or “remove” means that separating a clinical sample (e.g., into fractions) and reducing or eliminating one or more PPs. Removing need not mean 100% separation and reduction of one or more PPs. In some embodiments, removing is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%,53%, 52%, 51%, 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, or 40% separation and reduction of one or more PPs.

[0049] Removing may include separating a supernatant (e.g., from a pellet). Separating a supernatant typically involves the separation of a liquid (e.g., comprising 1-10 PPs) from solid particles (e.g., the pellet containing human cells and microbial cells) that have settled at the bottom of a container. The supernatant can be separated by decanting the supernatant into a new container or by pipetting (e.g., using a Pasteur pipette or a micropipette).

[0050] In some embodiments, removing may include filtration, whereby a sample is passed through a filter with a specific pore size that retains one or more microbial species (e.g., bacteria, virus, fungus, or parasite) while allowing other materials to pass through. In some embodiments, removing may be performed using a combination of centrifugation, filtration, and / or separating a supernatant.

[0051] In some embodiments, removing may include nucleic acid (e.g., DNA or RNA) isolation. Nucleic acid isolation, (also known as nucleic acid extraction), is a technique used to obtain DNA or RNA from a biological sample. The DNA or RNA that is isolated can be derived from the subject (e.g., a human subject) or from microbial species (e.g., bacteria, viruses, fungi, or parasites). Methods of performing nucleic acid isolation are known to those of skill in the art and can include, but are not limited to, phenol-chloroform extraction, silica- based spin columns, magnetic bead-based purification, organic solvent precipitation, solidphase extraction, and ultracentrifugation.Lysing cells

[0052] Methods provided herein include lysing cells (e.g., human cells, microbial cells) in a clinical sample (e.g., blood or urine). The term “lysing”, as used herein, refers to the breaking down or rupturing of a cell’s membranes to release its contents (e.g., DNA, RNA, proteins). In some embodiments, the subject (e.g., human) and microbial (e.g., bacteria, virus, or fungus) cells can be differentially lysed and fractioned based on their specific properties, such as size, density, or other chemical properties, including cell membrane or cell wall composition. There are several methods for performing cell lysis known in the art, such as mechanical cell lysis (e.g., homogenization), chemical cell lysis (e.g., detergent lysis, such as with Triton X-100 or sodium dodecyl sulfate (SDS)), enzymatic cell lysis (e.g., usinglysozyme), freeze-thaw lysis, ultrasonication, high-pressure homogenization, osmotic shock, or electroporation.

[0053] In some embodiments, methods provided herein include lysing cells (e.g., human cells, microbial cells). In some embodiments, methods provided herein include lysing cells (e.g., human cells, microbial cells) in a fraction (e.g., a first fraction) in the presence of a first detergent. Detergents are chemical compounds containing amphiphilic molecules that enable the disruption of cell membranes due to their ability to interact with and solubilize molecules or structures that are not water soluble. In some embodiments, the detergent is a nonionic detergent. A nonionic detergent is a detergent that does not carry a net electrical charge and is typically milder than ionic detergents. Milder means that a nonionic detergent is not as strong and will not disrupt cell membranes to the same degree as an ionic detergent. Non-limiting examples of nonionic detergents include Triton X-100 and NP-40. In some embodiments, the detergent is an ionic detergent. Ionic detergents are generally harsher than nonionic detergents. This class of detergents can be further subdivided into anionic detergents (e.g., sodium dodecyl sulfate (SDS), ammonium dodecyl sulfate (ADS), sodium lauryl sulfate (SLS), cetylpyridinium chloride), cationic detergents (e.g., cetyltrimethylammonium bromide (CTAB), benzyldimethyloctylammonium chloride), and zwitterionic detergents (e.g., CHAPS, pentaerythrityl palmitate). Other detergents known in the art are contemplated.

[0054] In some embodiments, methods provided herein include lysing cells (e.g., human cells, microbial cells) in a fraction (e.g., a first fraction or a second fraction) in the presence of a second detergent and a surfactant. A surfactant is a compound used to alter the surface properties of liquids and can facilitate the disruption of cell membranes due to their ability to solubilize lipids and prevent the clumping and / or aggregation of cellular material. In some embodiments, the presence of a surfactant protects non-human cells (e.g., cells of one or more microbial species) from lysis. In some embodiments, the presence of a surfactant does not protect non-human cells (e.g., cells of one or more microbial species) from lysis. Non-limiting examples of surfactants include Tween-20 (polysorbate-20), Tween-80 (polysorbate-80), NP-40, and Triton X-100. Other surfactants known in the art are contemplated.Amplification

[0055] In some embodiments, methods of the present disclosure include amplifying microbial DNA from lysed microbial species. Amplifying DNA means that the amount ofDNA (e.g., in a fraction) is increased by an enzymatic reaction. Non-limiting examples of methods for amplifying microbial DNA include polymerase chain reaction (PCR), quantitative PCR (qPCR), reverse-transcriptase PCR (RT-PCR), degenerate oligonucleotide PCR, primer extension pre-amplification, strand displacement amplification (SDA), helicase dependent amplification (HAD), transcription mediated amplification (TMA), recombinase polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP), whole genome amplification (WGA), multiple displacement amplification (MDA), random amplification of polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP), and rolling circle amplification (RCA). In some embodiments, whole genome microbial DNA from one or more lysed microbial species is amplified using MDA.

[0056] In some methods provided herein, the amount of amplified microbial DNA is increased 2-fold - 200-fold compared to control (e.g., as a result of adding a polyanionic polymer to a clinical sample). In some embodiments, the amount of amplified microbial DNA is increased 5-fold - 190-fold, 10-fold - 180-fold, 20-fold - 170-fold, 30-fold - 160- fold, 40-fold - 150-fold, 50-fold - 140-fold, 60-fold - 130-fold, 70-fold - 120-fold, 80-fold - 110-fold, or 90-fold - 100-fold compared to control. In some embodiments, the amount of amplified microbial DNA is increased 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30- fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, 100-fold, 105-fold, 110-fold, 115-fold, 120-fold, 125-fold, 130- fold, 135-fold, 140-fold, 145-fold, 150-fold, 155-fold, 160-fold, 165-fold, 170-fold, 175-fold, 180-fold, 185-fold, 190-fold, 195-fold, or 200-fold compared to control.

[0057] In some methods provided herein, the amount of amplified microbial DNA is increased 25% - 1000% compared to control (e.g., as a result of adding a polyanionic polymer to a clinical sample). In some embodiments, the amount of amplified microbial DNA is increased 50% - 950%, 100% - 900%, 150% - 850%, 200% - 800%, 250% - 750%, 300% - 700%, 350% - 650%, 400% - 600%, or 450% - 550% compared to control. In some embodiments, the amount of amplified microbial DNA is increased 25%, 50%, 75%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, 300%, 325%, 350%, 375%, 400%, 425%, 450%, 475%, 500%, 525%, 550%, 575%, 600%, 625%, 650%, 675%, 700%, 725%, 750%, 775%, 800%, 825%, 850%, 875%, 900%, 925%, 950%, 975%, or 1000% or more compared to control.

[0058] A control may be a clinical sample that has one or more PPs at a lower concentration than a clinical sample provided herein. A control may have 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%,29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%,45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% lower concentration of one or more PPs than a clinical sample provided herein.Identification

[0059] Methods provided herein include identifying one or more microbial species. The term “identifying” refers to confirming the presence of one or more microbial species (e.g., bacteria, virus, fungus, or parasite) in a sample (e.g., clinical sample).

[0060] In some embodiments, amplified DNA (e.g., microbial DNA) is identified. As used herein, identification of amplified DNA (e.g., microbial DNA) refers to confirmation of the presence of specific DNA sequences (occurring in a specific microbe) in a sample following DNA amplification. The identification can be qualitative, to confirm the presence or absence of the target DNA sequence, or quantitative, to provide information about the amount or concentration of the amplified DNA. Methods of detecting amplified DNA (e.g., microbial DNA) include, but are not limited to, agarose gel electrophoresis, polyacrylamide gel electrophoresis, ethidium bromide staining, SYBR green staining, fluorescent probes (e.g., TaqMan probes, molecular beacons, dual-labeled probes), DNA intercalating dyes (e.g., EvaGreen or SYBR Safe), DNA fragment analysis (e.g., capillary electrophoresis), real-time PCR (qPCR), digital PCR, nucleic acid hybridization (e.g., microarrays), enzyme-linked immunosorbent assays (ELISA), microscopy, or next-generation sequencing (e.g., Illumina sequencing or Nanopore sequencing).

[0061] In some embodiments, one or more microbial species are detected using sequencing (e.g., next-generation sequencing (NGS)). Sequencing data from clinical samples is analyzed to measure the breadth of coverage of a microbial species genome in the sequencing data. This approach is particularly advantageous in sequencing data obtained from clinical samples, which may include human DNA and other confounding DNA sources in addition to DNA from one or more microbial species. By measuring the breadth of coverage of a microbial species genome, contaminating DNA (e.g., human DNA, confounding DNA) may be removed from microbial DNA for processing. In some embodiments, only microbial species with a 0.5x - lOx microbial genome coverage areidentified in the present disclosure. In some embodiments, only microbial species with a 0.5x, 0.6x, 0.7x, 0.8x, 0.9x, l.Ox, l. lx, 1.2x, 1.3x, 1.4x, 1.5x, 1.6x, 1.7x, 1.8x, 1.9x, 2. Ox, 2. lx, 2.2x, 2.3x, 2.4x, 2.5x, 2.6x, 2.7x, 2.8x, 2.9x, 3.0x, 3. lx, 3.2x, 3.3x, 3.4x, 3.5x, 3.6x, 3.7x, 3.8x, 3.9x, 4. Ox, 4. lx, 4.2x, 4.3x, 4.4x, 4.5x, 4.6x, 4.7x, 4.8x, 4.9x, 5. Ox, 5. lx, 5.2x,5.3x, 5.4x, 5.5x, 5.6x, 5.7x, 5.8x, 5.9x, 6. Ox, 6. lx, 6.2x, 6.3x, 6.4x, 6.5x, 6.6x, 6.7x, 6.8x,6.9x, 7. Ox, 7. lx, 7.2x, 7.3x, 7.4x, 7.5x, 7.6x, 7.7x, 7.8x, 7.9x, 8.0x, 8. lx, 8.2x, 8.3x, 8.4x,8.5x, 8.6x, 8.7x, 8.8x, 8.9x, 9. Ox, 9. lx, 9.2x, 9.3x, 9.4x, 9.5x, 9.6x, 9.7x, 9.8x, 9.9x, or 10. Ox or more microbial genome coverage are identified in the present disclosure.

[0062] Sequencing reads following processing of a clinical sample are aligned to reference genomes from one or more microbial species. This alignment may be performed manually or by using an open-source software tool (e.g., Microbe-ID, DAMIAN, MIST, IDseq, mothur). The breadth of coverage of a microbial species may be calculated by simply counting sequencing reads for each microbial species or by reconstructing the whole genome of the one or more microbial species from sequencing reads in the clinical sample.Reconstructing the whole genome of one or more microbial species may be performed using an open-source software tool (e.g., flye, Canu, Raven, Shasta, Miniasm) or manually. Once a whole genome of one or more microbial species is reconstructed, the one or more microbial species may be identified using an open-source software tool (e.g., Kraken) or manually.

[0063] In some embodiments, where amplified microbial DNAis detected using a sequencing modality (e.g., Illumina sequencing or Nanopore sequencing), the genome coverage of the amplified microbial DNA is at least 0.8x, at least 0.9x, at least l.Ox, at least 1.5x, at least 2. Ox, at least 2.5x, at least 3. Ox, at least 3.5x, at least 4. Ox, at least 4.5x, or at least 5. Ox or more. In some embodiments, where amplified microbial DNAis detected using a sequencing modality (e.g., Illumina sequencing or Nanopore sequencing), the genome coverage of the amplified microbial DNA is 0.8x - 0.9x, 0.8x - l.Ox, 0.8x -1.5x, 0.8x - 2. Ox, 0.8x - 2.5x, 0.8x - 3. Ox, 0.8x - 3.5x, 0.8x - 4. Ox, 0.8x - 4.5x, 0.8x - 5. Ox, 0.9x - l.Ox, 0.9x - 1.5x, 0.9x - 2.0x, 0.9x - 2.5x, 0.9x - 3.0x, 0.9x - 3.5x, 0.9x - 4.0x, 0.9x - 4.5x, 0.9x - 5.0x, l.Ox - 1.5x, l.Ox - 2. Ox, l.Ox - 2.5x, l.Ox - 3. Ox, l.Ox - 3.5x, l.Ox - 4. Ox, l.Ox - 4.5x, l.Ox - 5. Ox, 1.5x - 2.0x, 1.5x - 2.5x, 1.5x - 3.0x, 1.5x - 3.5x, 1.5x - 4.0x, 1.5x - 4.5x, 1.5x - 5.0x, 2. Ox - 2.5x, 2. Ox - 3. Ox, 2. Ox - 3.5x, 2. Ox - 4. Ox, 2. Ox - 4.5x, 2. Ox - 5. Ox, 2.5x - 3. Ox, 2.5x - 3.5x, 2.5x - 4. Ox, 2.5x - 4.5x, 2.5x - 5. Ox, 3. Ox - 3.5x, 3. Ox - 4. Ox, 3. Ox - 4.5x, 3. Ox - 5. Ox, 3.5x - 4. Ox, 3.5x - 4.5x, 3.5x - 5. Ox, 4. Ox - 4.5x, 4. Ox - 5. Ox, or 4.5x - 5. Ox.Microbial Species

[0064] Methods provided herein include enriching, identifying and / or quantifying one or more microbial species in a clinical sample. The term “microbial species”, as used herein, is any microorganism that is present in a sample (e.g., a clinical sample). In some embodiments, the microbial species is a bacteria, a virus, a fungus, a parasite, or any combination thereof. The microbial species present in a sample may be a commensal organism, which is a microorganism (e.g., bacteria, virus, fungus, or parasite) that lives in or on a subject without causing harm to the host or providing any significant benefits, or may be a pathogenic organism, which is a microorganism (e.g., bacteria, virus, fungus, mold, or parasite) that has disease-causing potential. The terms “pathogenic organism” or “pathogenic microbial species”, are used interchangeably herein and refer to a microorganism (e.g., bacteria, virus, fungus, or parasite), that has the ability to cause disease in a subject by invading, colonizing, and / or multiplying within the subject, leading to various illnesses or infections. In some embodiments, a commensal organism can turn into a pathogenic organism in a process referred to as “commensal -to-pathogen transition”. Non-limiting factors contributing to this shift include: i) genetic mutations that may result in the acquisition of new genes or the loss of genes that regulate virulence, increasing the organism’s pathogenic potential; ii) horizontal gene transfer, in which a commensal organism acquires virulence factors or antibiotic resistance genes from another microorganism; iii) changes in environmental conditions, such as antibiotic use or immune system suppression; and, iv) microbiota dysbiosis, in which alterations in a host’s microbiome composition and diversity creates opportunities for commensal organisms to complete with or displace other microorganisms.

[0065] In some embodiments, one or more microbial species in a clinical sample provided herein is a pathogenic microbial species. One microbial species or multiple microbial species in a clinical sample may be pathogenic. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more microbial species are pathogenic microbial species.

[0066] In some embodiments, a pathogenic microbial species is a virus. In some embodiments, a pathogenic microbial species is a fungus. In some embodiments, a pathogenic microbial species is a parasite.Sample

[0067] In some embodiments, a method for removing a PP form a clinical sample comprises obtaining a clinical sample comprising one or more microbial species from a subject. The term “obtaining”, as used herein, refers to collecting a clinical sample (e.g., blood or urine) from a subject at a designated time. A clinical sample is a biological sample obtained from a subject. Non-limiting examples of clinical samples include a blood sample (e.g., whole blood, plasma, or serum), a urine sample, a saliva sample, a stool sample, a cerebrospinal fluid sample, a throat swab sample, an oral swab sample, a bronchial lavage sample. In some embodiments, a clinical sample is a blood sample, wherein the blood sample is a whole blood sample, a plasma sample, or a serum sample. In some embodiments, the blood sample is obtained from a subject by venipuncture or finger prick. In some embodiments, a clinical sample is a urine sample, wherein the urine sample is obtained by midstream urine collection, catheterization (e.g., using a Foley catheter), or bladder puncture. A clinical sample may, in some embodiments, contain low levels of commensal bacteria from the skin and / or urinary tract microbiome.Subjects

[0068] Methods provided herein include obtaining a clinical sample from a subject. A subject may be any organism that can be infected by a microbial species. In some embodiments, the subject is a mammal. In some embodiments, the subject is a non-human primate (e.g., a laboratory animal such as a rhesus monkey). In some embodiments, the subject is a rodent, such as a rat or mouse. In some embodiments, the subject is a goat, rabbit, sheep, or pig. In some embodiments, the subject is a human.

[0069] Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the reminder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein.EXAMPLESExample 1. Sodium polyanethol sulfonate (SPS) binds serum / plasma proteins

[0070] Sodium polyanethol sulfonate (SPS) is a polyanionic polymer widely used as an anticoagulant in blood collection containers. There are several advantages for using SPS in blood collection, such as preventing lysis of bacterial cells by the complement system, reduction of blood cell clumping, and facilitation of the separation of blood components into solids (e.g., red blood cells and white blood cells, etc.) and liquids (e.g., plasma, small molecular weight nucleic acid amplification inhibitors, etc.). Despite these benefits, persistence of SPS in the liquid phase can block downstream processes such as nucleic acid amplification. Much like DNA, SPS is soluble in water, but not in alcohol, thus it tends to copurify with DNA during whole genome amplification processes. In order to effectively amplify pathogen genomes directly from whole human blood, removal of SPS from the molecular reactions during the genome amplification, or mitigation of its presence during sample processing is imperative.

[0071] A possible explanation for SPS remaining after splitting blood into cells and plasma is that it forms large complexes with other proteins in the liquid phase and coprecipitates during the separation. To test this, a sample with 10 mg / mL lysozyme was treated with 0.05% SPS, which resulted in immediate precipitation with a visible pellet (Fig. 1A). To remove the soluble SPS in the supernatant fraction after centrifugation, the precipitate is then treated with high concentration of SPS, after which it no longer pellets (Fig. IB). This demonstrates that addition of high concentration of SPS to human blood samples collected for microbial genome amplification could disrupt SPS-protein aggregation and reduce unwanted effects of SPS on microbial DNA amplification.Example 2: Addition of high concentration SPS improves sequencing outputs

[0072] To examine the effect of high concentration SPS as a precipitate mitigation strategy on microbial DNA amplification efficiency, ImL of whole blood samples from a healthy donor were collected in vacuum liquid containers containing SPS and diluted to a final volume of 3mL with lx DPBS (Ca+2and Mg+2free, ThermoFisher, Waltham, MA). The samples were spiked with 8.8 CFU / mL of S. aureus to simulate bacteremic blood samples.

[0073] Each sample was treated with 0%, 0.5%, 1%, or 5% SPS. Clinical sample processing was performed to remove human cells and concentrate the remaining bacterialcells. These bacterial cells were lysed at 37°C. Bacterial DNA from lysed bacterial cells was concentrated and subjected to REPLI-g Single Cell MDA. Total amplified DNA was quantified using the Qubit dsDNAHS Assay kit (ThermoFisher, Waltham, MA).

[0074] The concentration of double-stranded DNA (dsDNA) and total megabase (MB) count were measured. dsDNA concentration in the blood samples treated with 0.5%, 1% or 5% SPS was increased by at least 100-fold compared to a 0% SPS control sample (Figs. 2A and 2B). Bacterial whole genome coverage values in blood samples treated with 0.5%, 1% and 5% SPS are significantly high as compared to 0% SPS control (Fig. 2C).

[0075] Ethylenediaminetetraacetic acid (EDTA), heparin, and anticoagulant citrate dextrose solution (ACD-A) are other commonly-used anticoagulants. Clinical blood sample collection tubes containing EDTA, heparin, and ACD-A were tested for efficacy in preservation of intact microbial cells and extraction of DNA for amplification. Whole blood samples from a healthy donor were collected in vacuum liquid containers containing SPS, ACD-A, EDTA, or heparin. The experimental samples were processed as described above. Fig. 3 shows that high concentration SPS produces significantly increased microbial DNA identification using SPS containing vacutainers as compared to ACD-A, EDTA or heparin containing vacutainers.Together these data demonstrate that treating whole blood clinical samples collected in SPS vacutainers with high concentration of SPS ensures preservation of intact microbial cells and at the same time reduces the negative impact of SPS on downstream molecular reactions to amplify enriched bacterial DNA.

Claims

CLAIMSWhat is claimed is:

1. A method for removing a polyanionic polymer from a clinical sample, the method comprising:(i) obtaining a clinical sample from a subject comprising one or more microbial species in a container comprising 0.01% - 1.00% of a first polyanionic polymer;(ii) adding between 0.1% - 10% of a second polyanionic polymer to the clinical sample; and(iii) removing from the clinical sample from step (ii) a first fraction comprising the first polyanionic polymer and the second polyanionic polymer.

2. A method for amplifying microbial DNA in a clinical sample collected from blood, the method comprising:(i) obtaining a clinical sample from a subject comprising one or more microbial species in a container comprising 0.01% - 1.00% of a first polyanionic polymer;(ii) adding between 0.1% - 10% of a second polyanionic polymer to the clinical sample;(iii) removing from the clinical sample from step (ii) a first fraction comprising the one or more microbial species and cells from the subject and a second fraction comprising the first polyanionic polymer and the second polyanionic polymer;(iv) lysing the one or more microbial species and cells from the subject from the first fraction; and(v) amplifying microbial DNA from the lysed microbial species.

3. The method of claim 2, further comprising (vi) identifying the one or more microbial species from the amplified microbial DNA.

4. The method of claim 2 or claim 3, wherein adding the second polyanionic polymer increases the amount of amplified microbial DNA at least 5-fold compared to control.

5. The method of any one of claims 2-4, wherein the amplifying is whole genome amplification.

6. The method of any one of claims 1-5, wherein the first polyanionic polymer is sodium polyanethol sulfonate (SPS).

7. The method of any one of claims 1-5 wherein the second polyanionic polymer is SPS.

8. The method of any one of claims 1-5, wherein the first polyanionic polymer is SPS and the second polyanionic polymer is SPS.

9. The method of any one of claims 1-5, wherein the 0.1% - 10% of the second polyanionic polymer is added to a clinical sample comprising blood.

10. The method of any one of claims 1-9, wherein the subject is a human.

11. The method of any one of claims 1-10, wherein the microbe is a pathogenic microbe.

12. A method of removing or reducing the amount of sodium polyanethol sulfonate (SPS) in a clinical sample containing nucleic acids, the method comprising:(a) adding SPS to the clinical sample,(b) solubilizing SPS-protein precipitates; and(c) removing the soluble fraction comprising SPS.

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